Methods for treating autism spectrum disorder and associated symptoms

ABSTRACT

The present disclosure relates to compositions and methods for treating autism spectrum disorder (ASD) by restoring an ASD patient&#39;s gut microbiota. These methods can be used with ASD patient with or without ongoing gastrointestinal symptoms. Provided here is a method for modulating the abundance of one or more gut microorganisms in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition comprising a fecal microbe preparation to achieve at least a 2-fold change of the abundance of said one or more fecal microorganisms, wherein said one or more gut microorganisms are selected from the group consisting of Bifidobacterium, Prevotella, Desulfovibrio, Copprococcus, Clostridium, and Bacteroides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage of International ApplicationNo. PCT/US2017/037499, filed Jun. 14, 2017, which claims priority toU.S. Provisional Application No. 62/350,693, filed Jun. 15, 2016, bothof which are herein incorporated by reference in their entireties.

BACKGROUND

The present disclosure relates to methods of modulating gut microbiomeand methods for treating autism spectrum disorder (ASD). Autism spectrumdisorder (ASD) is a complex neurodevelopmental condition characterizedby widespread abnormalities of social interactions and communication, aswell as restricted interests and repetitive behaviors. ASD typicallyappears during the first three years of life and manifests incharacteristic symptoms or behavioral traits. A diagnosis of ASD nowincludes several conditions that used to be diagnosed separately:autistic disorder, pervasive developmental disorder not otherwisespecified (PDD-NOS), and Asperger syndrome. All of these conditions arenow encompassed by the diagnostic criteria for autism spectrum disorderas set forth in the American Psychiatric Association's Diagnostic &Statistical Manual of Mental Disorders, Fifth Edition (DSM-V).

In addition to the spectrum of symptoms seen within these principaldiagnostic criteria, ASD individuals display a wide range ofneurological comorbidities, including intellectual disability, epilepsy,and anxiety and mood disorders, as well as non-neurologicalcomorbidities, including blood hyperserotonemia, immune dysregulation,and GI dysfunction (e.g., chronic constipation, diarrhea, abdominalpain, and gastroesophageal reflux).

To date, there are no FDA-approved treatments for reducing oreliminating the core symptoms of autism spectrum disorder. The only twomedications approved by the FDA for treating autism, risperidone (soldunder Risperdal®) and aripiprazole (sold under Abilify®), arespecifically indicated for reducing irritability in subjects having ASD.Accordingly, there remains a need in the art for improved methods fortreating and reducing the severity and incidence of symptoms associatedwith autism spectrum disorder. This application provides a method fortreating an ASD patient (with or without a GI symptom) by transferringbeneficial fecal bacteria to replace, restore, or rebalance the ASDpatient's gut microbiota, a treatment referred to here as MicrobiotaTransfer Therapy (MTT). This application further provides a method fortreating an ASD in a patient by selectively modulating the abundance ofcertain fecal microbes (e.g., bacteria) or bacteriophages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents Gastrointestinal Symptom Rating Scale (GSRS) data fortrial participants.

FIG. 2 presents GSRS subscale data collected prior to (“pre”) andfollowing (“post”) MTT treatment.

FIG. 3 presents continuous improvements of both average GSRS and averagePGI-R scores of the participants.

FIG. 4 presents Aberrant Behavior Checklist (ABC) data for MTT trialparticipants.

FIG. 5 presents GSRS scores collected 8 weeks post-treatment.

FIG. 6 is a graph demonstrating the lack of correlation between age andthe degree of CARS score improvement.

FIG. 7 is a graph demonstrating that end-of-treatment PGI-R scores hadlittle correlation with age.

FIG. 8 is a graph demonstrating the lack of correlation between thedegree of improvement on CARS and initial GSRS score.

FIG. 9 (including panels a to e) describes the improvement of GI- andcore ASD-related symptoms of 18 ASD-afflicted children treated with MTT.Children are treated with vancomycin for two weeks followed by theadministering of a fecal bacteria composition for 8 weeks, with a singlefollow-up evaluation 8 weeks after treatment ended. Panel a. Changes inGSRS scores. GSRS is scored on a Likert scale from 1 (no symptoms) to 7(very severe discomfort). Panel b. Changes in PGI-R scores (Overallautism/related symptoms). PGI-R is scored from −3 (much worse), −2(worse), −1 (slightly worse), 0 (no change), 1 (slightly better), 2(better) to 3 (much better) compared to baseline. Panel c. CARSassessment pre-treatment, post treatment and 8 weeks post treatment.Panel d. Total SRS score pre-treatment, post treatment and 8 weeks posttreatment. Panel e. Total ABC score pre-treatment, post treatment and 8weeks post treatment. The data points represent 18 individualparticipants, and some data points overlap in the box plot. Asterisks(at the top of the box-plot) indicate whether individuals (at each timepoints) significantly decrease since pre-treatment (Week 0). ns:not-significant, *: p<0.05, **: p<0.01, ***: p<0.001 (two-tailed pairedt-test). Two participants who had less than 50% improvement in GSRSscores are defined as non-responders and color-coded in grey.

FIG. 10 (including panels a to c) provides a breakdown of GSRScomponents and improvements in patients. Panel a. GSRS subscores atbaseline, MTT treatment end, and 8 weeks after treatment. Panel b.Results of daily stool records, averaged over 2 weeks. Panel c.Subscales of the ABC vs. time. *: p<0.05, **: p<0.01, ***: p<0.001(two-tailed paired t-test).

FIG. 11 demonstrates a correlation between GSRS and PGI-R (based on thedata shown in FIG. 10, panels a and b). The Pearson correlation testshowed r=−0.56 and p<0.001.

FIG. 12 shows Vineland Developmental Age (in years) for differentsubscales and for the average of all the subscales, measured at baselineand at the end of observation 4 months later. Note that the averagechronological age is 10.9 years at the start of treatment, so atbaseline there are delays in all areas, especially in the core autismareas of language and social (interpersonal) ability. *: p<0.05, **:p<0.01, ***: p<0.001 (two-tailed paired t-test).

FIG. 13 shows subscores of the PGI-R at end of treatment (week 10). Thescale goes from 3 (much better) to 2 (better) to 1 (slightly better) to0 (no change) to minus 3 (much worse). Scores are similar after 8 weeksof no treatment (week 18). The data points represent 18 individualparticipants, and some data points overlap in the box plot.

FIG. 14 (including panels a to h) describes stool microbiota changes inASD patients receiving MTT. Panel a. Changes in Faith's PhylogeneticDiversity (PD) in the ASD microbiota as measured from stool samples.Orange line indicates median PD of the donor samples, and green lineindicates median PD of neurotypical controls at week 0. Panel b. Changein Faith PD tracked on a per individual basis for all MTT recipients.Most individuals experienced an increase in gut microbiota PD. Panel c.Unweighted UniFrac distances between ASD gut microbiota and mostrelevant donor sample (initial donor sample at weeks 0 and 3, mostrecent maintenance dose sample at weeks 10 and 18). Green line indicatesthe median interpersonal variation between neurotypical controls andillustrates that prior to treatment the difference in gut microbiotacomposition between MTT recipients and donors is on the order of normalinterpersonal variation. Following treatment, the MTT recipients aremore similar to donors than normal interpersonal variation. Panel d.Distances between ASD gut microbiota and donor sample on a perindividual basis. Most individuals become more similar to the donor overthe study period. Panels e to h. Boxplots illustrating abundance of fourgenera, Bifidobacterium, Prevotella, Desulfovibrio, and Bacteroides, inthe gut microbiota by group (top), and change in abundance 8 weeks afterMTT in the ASD group (bottom).

FIG. 15 (including panels a and b) describes stool microbiota changes inASD patients received MTT and further classified according to the oralor rectal administration of MIVIT. Panel a. Changes in Faith'sPhylogenetic Diversity (PD) in the ASD microbiota as measured from stoolsamples. Orange line indicates median PD of the donor samples, and greenline indicates median PD of neurotypical controls at week 0. Panel b.Unweighted UniFrac distances between ASD gut microbiota and mostrelevant donor sample (initial donor sample at weeks 0 and 3, mostrecent maintenance dose sample at weeks 10 and 18). Green line indicatesthe median interpersonal variation between neurotypical controls andillustrates that prior to treatment the difference in gut microbiotacomposition between MTT recipients and donors is on the order of normalinterpersonal variation. Following treatment, the MTT recipients aremore similar to donors than normal interpersonal variation.

FIG. 16 presents Change in community richness, as measure with anon-phylogenetic diversity metric.

FIG. 17 describes engraftment plots with four diversity metrics.

FIG. 18 (including panels a to d) describes microbiota analysis of fecalswab samples from ASD patients received MTT. Each of panels a to dcorrespond to panels a to d of FIG. 14, respectively. Panel a. Changesin Faith's Phylogenetic Diversity (PD) in the ASD microbiota (n=20).Orange lines indicates median PD of the donor samples (dashed linerepresents initial donor samples (n=4), and dashed line representsmaintenance dose samples (n=2)), and green line indicates median PD ofneurotypical controls at week 0 (n=18). Panel b. Change in Faith PDtracked on a per individual basis for all MTT recipients. Panel c.Unweighted UniFrac distances between ASD gut microbiota and mostrelevant donor sample (initial donor sample at weeks 0 and 3, mostrecent maintenance dose sample at weeks 10 and 18). Green line indicatesthe median interpersonal variation between neurotypical controls. Paneld. Distances between ASD gut microbiota and donor sample on a perindividual basis.

FIG. 19 (including panels a to d) describes stool virome changes in ASDpatients receiving MTT. Panel a. Diversity indices, Shannon's H' (ameasure of biodiversity and richness; right) and Peilou's J (a measureof evenness; left), of ASD participants. ASD diversity indices decreaseat week 3 following vancomycin and the initial high dose MMT treatmentand then rebound over time and stabilized following treatment withmaintenance doses of MMT. Fecal samples are collected at all four timepoints for four out of the 12 ASD subjects where the bacteriophagecommunities are assessed. The responders (indicated by a grey line)rebounded in biodiversity, richness and evenness following FMT. Incontrast, the non-responder (indicated by a red line) does not recover.Panel b. Nonmetric multidimensional scaling of Bray-Curtis dissimilarity(right; 2D-stress=0.2467) and Jaccard (left; 2D-stress=0.2212) distancesreveal that ASD gut bacteriophage communities are more similar to donorgut bacteriophage communities following both the high and low MTT doses.Panel c, Analyses of ASD virome composition at week 10 shows engraftmentof donor bacteriophage populations across all ASD subjects. In >80% ofthe subjects, the starting (week 0) bacteriophage populations make up<20% of the virome at week 10. Panel d. In contrast, analyses of viromecomposition of the untreated neurotypical children at week 18 reveals ahigh percentage (>60%) of starting (week 0) bacteriophage populations.

FIG. 20 provides a neighbor-joining phylogenetic tree of 16S rRNA genesequences of the type strains of Prevotella species deposited in GenBankto show the relationships of strains within the genus. Bootstrap values(expressed as percentages of 1000 replicates) are shown at the nodes.The numbers that precede each strain are the GenBank accession numbers,while those that follow the species names are the Culture Collectionnumbers. The type species of Bacteroides (Bacteroides fragilis, NCTC9343) and Porphyromonas (Porphyromonas asaccharolytica, ATCC 25260),both former members of the same genus, and Escherichia coli (K12) areincluded for comparison.

FIG. 21 describes phylogenetic relationships based on 16S rRNA genesequence analyses of the taxa within the order Desulfovibrionales forwhich 16S rRNA gene sequences are available. The scale bar represents 10inferred nucleotide substitutions per 100 nucleotides. The dendrogram isreconstructed from distance matrices using the neighbor-j oining method.

FIG. 22 describes a phylogenetic tree constructed from partial 16S rRNAgene sequences of members of the family Bifidobacteriaceae. The tree isrooted with Escherichia coli, and constructed by using theneighbor-joining method with bootstrap values calculated from 1000 trees(represented as percentages and given at each branch). For each species,the GenBank accession number for the respective 16S rRNA gene sequenceis indicated. The scale bar shows the number of nucleotide substitutionsper site.

FIG. 23 (including panels a to c) provides a breakdown of GSRScomponents and improvements in patients. GSRS average scores of all,Panel a. and individual participants, Panel b. at baseline, MTTtreatment end (week 10), 8 weeks after treatment, and 2-years aftertreatment (follow-up). Panel c. Results of average GSRS subscale scorespatient at baseline, MTT treatment end (week 10), 8 weeks aftertreatment (week 18), and 2-years after treatment (follow-up). Analysisfor 2-year follow-up is between 1.7 and 2.1 years after end oftreatment.

FIG. 24 provides the results of daily stool records, averaged over 2weeks. Type 1 stool: Separate hard lumps, like nuts (hard to pass); Type2: Sausage-shaped, but lumpy; Type 6: Fluffy pieces with ragged edges, amushy stool; and Type 7: Watery, no solid pieces, entirely liquid.

FIG. 25 (including panels a and b) provides the scores of the PGI-R atend of treatment (week 10), 8 weeks after treatment (week 18), and atthe 2-year follow-up. The scale goes from 3 (much better) to 2 (better)to 1 (slightly better) to 0 (no change). Scores are similar after 8weeks of no treatment (week 18) and 2-years of no treatment (Follow-up).The data points represent 17 individual participants.

FIG. 26 (including panels a and b) shows Vineland Developmental Age inyears (not including motor skills) for Panel a. different subscales andfor Panel b. the average of all the subscales, measured at baseline, 8weeks after treatment, and 2-years after treatment (Follow-up). Notethat the average initial developmental age is 5.57 years vs. achronological age of 10.9 years, so at baseline there are delays in allareas, especially in the core autism areas of language and social(interpersonal) ability.

FIG. 27 (including panels a and b) shows SRS subscales scores atbaseline, MTT treatment end (week 10), 8 weeks after treatment, and2-years after treatment (follow-up). Panel b, shows average overall SRSscores. The scores range from severe range, to mild-to-moderate range,to normal range. The data points represent 17 individual participants.

FIG. 28 (including panels a and b) shows ABC subscales scores atbaseline, MTT treatment end (week 10), 8 weeks after treatment, and2-years after treatment (follow-up). Panel b, shows average overall ABCscores. The data points represent 18 individual participants.

FIG. 29 (including panels a to d) show total CARS scores at baseline,MTT treatment end (week 10), 8 weeks after treatment, and 2-years aftertreatment (follow-up). The scores range from severe symptoms, tomild-to-moderate symptoms, to minimal-to-no symptoms. Panel b. shows thepercentage of participants with severe symptoms, mild-to-moderatesymptoms, and minimal-to-no symptoms, at baseline, end of treatment, andfollow-up. The data points represent 18 individual participants. Panelsc and d. demonstrate a correlation between GSRS and CARS based onpercentage change and difference.

FIG. 30 (including panels a to d) describes stool microbiota changes inASD patients receiving MTT, as measured with an observed OTU diversitymetric, Panel a. and Faith's Phylogenetic Diversity (PD), Panel b. Mostindividuals experienced an increase in gut microbiota PD. Panel c, showsMann-Whitney test of diversity comparisons 2-years after treatmentbetween participants receiving oral and rectal administration of MTT.Panel d. shows a Mann-Whitney test of diversity comparisons 2-yearsafter treatment between male and female participants. ns: notsignificant, *: p<0.05, **: p<0.01 (two-tailed Wilcoxon signed-ranktest).

FIG. 31 (including panels a to c) shows boxplots illustrating abundanceof three genera, Bifidobacterium (Panel a.), Prevotella (Panel b.), andDesulfovibrio (Panel c.), in the gut microbiota, and change in abundanceat week 3 during treatment, week 10 at treatment end, 8 weeks after notreatment, and 2-years after no treatment (Follow-up) in the ASDparticipants. ns: not significant, *: p<0.05, **: p<0.01 (two-tailedWilcoxon signed-rank test).

SUMMARY

In one aspect, this application provides a method for increasing theabundance of one or more gut microorganisms in a subject in needthereof, the method comprising treating the subject by administering atherapeutically effective amount of a pharmaceutical compositioncomprising a fecal microbe preparation, wherein the subject exhibits atleast a 2-fold increase of the abundance of the one or more fecalmicroorganisms after the treatment as compared to before initiating thetreatment, wherein the one or more gut microorganisms are from a genusselected from the group consisting of Bifidobacterium, Prevotella, andDesulfovibrio.

In another aspect, this application provides a method for increasing thephylogenetic diversity of fecal microbiota, fecal phage virome, or both,of a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a fecal microbe preparation, wherein the subjectexhibits at least a two-fold increase of fecal microbiota or fecal phagevirome diversity after the treatment as compared to before initiatingthe treatment.

In one aspect, this application provides a method comprising:determining the relative abundance of one or more gut microorganisms ina subject having an ASD or having an ASD sibling, and administering atherapeutically effective amount of a pharmaceutical compositioncomprising a fecal microbe preparation to achieve at least a 2-foldchange of the abundance of the one or more fecal microorganisms, whereinthe one or more gut microorganisms are from a genus selected from thegroup consisting of Clostridium, Bacteroides, Eggerthella,Bifidobacterium, Prevotella, and Desulfovibrio.

In another aspect, this application provides a method for modulating theabundance of one or more gut microorganisms in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising a fecal microbe preparation to achieve at least a 2-foldchange of the abundance of the one or more fecal microorganisms, whereinthe one or more gut microorganisms are selected from the groupconsisting of Bifidobacterium, Prevotella, Desulfovibrio, Copprococcus,Clostridium, Eggerthella, and Bacteroides.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application isspecifically and individually indicated to be incorporated by reference.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. As used herein, the term “substantially” as in, for example,the phrase “substantially all peptides of an array,” refers to at least90%, preferably at least 95%, more preferably at least 99%, and mostpreferably at least 99.9%, of the peptides of an array. Other uses ofthe term “substantially” involve an analogous definition.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the disclosure. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the disclosure, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the disclosure.

As used herein, the term “treating” refers to (i) completely orpartially inhibiting a disease, disorder or condition, for example,arresting its development; (ii) completely or partially relieving adisease, disorder or condition, for example, causing regression of thedisease, disorder and/or condition; or (iii) completely or partiallypreventing a disease, disorder or condition from occurring in a patientthat may be predisposed to the disease, disorder and/or condition, buthas not yet been diagnosed as having it. Similarly, “treatment” refersto both therapeutic treatment and prophylactic or preventative measures.In the context of autism spectrum disorder, “treat” and “treating”encompass alleviating, ameliorating, delaying the onset of, inhibitingthe progression of, or reducing the severity of one or more symptomsassociated with an autism spectrum disorder. In one aspect, treating canalso mean modulating the abundance of selected bacterial genera.

As used herein, a “subject” can be a human or animal including, but notlimited to, a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent,e.g., rats and mice, and primate, e.g., monkey. Preferred subjects arehuman subjects. The human subject may be a pediatric, adult or ageriatric subject.

As used herein, a “microbiota” and “flora” refer to a community ofmicrobes that live in or on a subject's body, both sustainably andtransiently, including eukaryotes, archaea, bacteria, and viruses(including bacterial viruses (i.e., phage)). A “fecal microbiota” or“fecal microbiota preparation” refers to a community of microbes presentin or prepared from a subject's feces. A non-selective fecal microbiotarefers to a community or mixture of fecal microbes derived from adonor's fecal sample without selection and substantially resemblingmicrobial constituents and population structure found in such fecalsample.

As used herein, “therapeutically effective amount” or “pharmaceuticallyactive dose” refers to an amount of a composition which is effective intreating the named disease, disorder or condition.

As used herein, “isolated” or “purified” refers to a bacterium or otherentity or substance that has been (1) separated from at least some ofthe components with which it is associated when initially produced(whether in nature or in an experimental setting), and/or (2) produced,prepared, purified, and/or manufactured by the hand of man. Isolated orpurified bacteria can be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, or more of the other components with which they are initiallyassociated.

As used herein, the terms “non-pathogenic” in reference to a bacteriumor any other organism or entity includes any such organism or entitythat is not capable of causing or affecting a disease, disorder orcondition of a host organism containing the organism or entity.

As used herein, “spore” or a population of “spores” includes bacteria(or other single-celled organisms) that are generally viable, moreresistant to environmental influences such as heat and bacteriocidalagents than vegetative forms of the same bacteria, and typically capableof germination and out-growth. “Spore-formers” or bacteria “capable offorming spores” are those bacteria containing the genes and othernecessary abilities to produce spores under suitable environmentalconditions.

As used herein, “colony forming units” (cfu) refers to an estimate ofthe number of viable microorganism cells in a given sample. The numberof cfu can be assessed by counting the number of colonies on an agarplate as in standard methods for determining the number of viablebacterial cells in a sample.

As used herein, “viable” means possessing the ability to multiply. Theviability of bacterial populations can be monitored as a function of themembrane integrity of the cell. Cells with a compromised membrane areconsidered to be dead or dying, whereas cells with an intact membraneare considered live. For example, SYTO 9 and propidium iodide are usedto stain and differentiate live and dead bacteria. See Stocks, CytometryA. 2004 October; 61(2):189-95. Cell viability can also be evaluated viamolecular viability analyses, e.g., a PCR-based approach, which candifferentiate nucleic acids associated with viable cells from thoseassociated with inactivated cells. See Cangelosi and Mescheke, ApplEnviron Microbiol. 2014 October; 80(19): 5884-5891.

As used herein, “Shannon Diversity Index” refers to a diversity indexthat accounts for abundance and evenness of species present in a givencommunity using the formula H=−Σ_(i=1) ^(R) p_(i) ln p_(i), where H isShannon Diversity Index, R is the total number of species in thecommunity, and p_(i) is the proportion of R made up of the ith species.Higher values indicate diverse and equally distributed communities, anda value of 0 indicates only one species is present in a given community.For further reference, see Shannon and Weaver, (1949) The mathematicaltheory of communication. The University of Illinois Press, Urbana. 117pp.

As used herein, “antibiotic” refers to a substance that is used to treatand/or prevent bacterial infection by killing bacteria, inhibiting thegrowth of bacteria, or reducing the viability of bacteria.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that ischaracterized by impairments in social interaction and communication,restricted interests, and repetitive behavior. Individuals on the autismspectrum experience widely varying degrees and types of impairments,from mild to severe. Although early detection and interventions areencouraged to maximize the benefits and reduce the severity of thesymptoms, individuals of any age can benefit from interventions andtherapies that can reduce symptoms and increase skills and abilities.Appropriate subjects for the methods described herein include, withoutlimitation, humans diagnosed as having or suspected of having autismspectrum disorder. In some cases, appropriate subjects for the methodsprovided herein are considered to be at increased risk (e.g., moderateor high risk) of developing ASD. In some cases, the subject has beendiagnosed as having a condition meeting diagnostic criteria for ASD asset forth in the DSM-V. In other cases, the subject has awell-established DSM-IV diagnosis of autistic disorder, Asperger'sdisorder, or pervasive developmental disorder not otherwise specified(PDD-NOS).

The methods provided herein result in, or are aimed at achieving adetectable improvement in one or more indicators or symptoms of ASDincluding, without limitation, including, but not limited to, changes ineye tracking, skin conductance and/or EEG measurements in response tovisual stimuli, difficulties engaging in and responding to socialinteraction, verbal and nonverbal communication problems, repetitivebehaviors, intellectual disability, difficulties in motor coordination,attention issues, sleep disturbances, and physical health issues such asgastrointestinal disturbances.

Several screening instruments are known in the art for evaluating asubject's social and communicative development and thus can be used asaids in screening for and detecting changes in the severity ofimpairment in communication skills, social interactions, and restricted,repetitive and stereotyped patterns of behavior characteristic of autismspectrum disorder. Evaluation can include neurologic and geneticassessment, along with in-depth cognitive and language testing.Additional measures developed specifically for diagnosing and assessingautism include the Autism Diagnosis Interview-Revised (ADI-R), theAutism Diagnostic Observation Schedule (ADOS-G) and the Childhood AutismRating Scale (CARS).

According to CARS, evaluators rate the subject on a scale from 1 to 4 ineach of 15 areas: Relating to People; Imitation; Emotional Response;Body Use; Object Use; Adaptation to Change; Visual Response; ListeningResponse; Taste, Smell, and Touch Response and Use; Fear; VerbalCommunication; Nonverbal Communication; Activity; Level and Consistencyof Intellectual Response; and General Impressions.

A second edition of CARS, known as the Childhood Autism Rating Scale—2or CARS-2, is developed by Schopler et al. (Childhood Autism RatingScale—Second edition (CARS2): Manual. Los Angeles: Western PsychologicalServices, 2010). The original CARS is developed primarily withindividuals with co-morbid intellectual functioning and is criticizedfor not accurately identifying higher functioning individuals with ASD.CARS-2 retained the original CARS form for use with younger or lowerfunctioning individuals (now renamed the CARS2-ST for “Standard Form”),but also includes a separate rating scale for use with higherfunctioning individuals (named the CARS2-HF for “High Functioning”) andan unscored information-gathering scale (“Questionnaire for Parents orCaregivers” or CARS2-QPC) that has utility for making CARS2ST andCARS2-HF ratings.

Another symptom rating instrument useful for assessing changes insymptom severity before, during, or following treatment according to amethod provided herein is the Aberrant Behavior Checklist (ABC). SeeAman et al., Psychometric characteristics of the aberrant behaviorchecklist. Am J Ment Defic. 1985 March; 89(5):492-502. The ABC is asymptom rating checklist used to assess and classify problem behaviorsof children and adults in a variety of settings. The ABC includes 58items that resolve onto five subscales: (1) irritability/agitation, (2)lethargy/social withdrawal, (3) stereotypic behavior, (4)hyperactivity/noncompliance, and (5) inappropriate speech.

The present inventors observed that autistic individuals, regardless ofthe presence or absence of comorbid gastrointestinal distress, havefewer species of gut bacteria as compared to neurotypical individuals.The present inventors also found that restoring the species diversity ofgut bacteria helps to treat autistic symptoms in patients in needthereof.

In one aspect, this application provides a method for increasing theabundance of one or more gut microorganisms in a subject in needthereof, the method comprising treating the subject by administering atherapeutically effective amount of a pharmaceutical compositioncomprising a fecal microbe preparation, wherein the subject exhibits atleast a 50-fold increase of the abundance of the one or more fecalmicroorganisms after the treatment as compared to before initiating thetreatment, wherein the one or more gut microorganisms are from a genusselected from the group consisting of Bifidobacterium, Prevotella, andDesulfovibrio. In one aspect, a subject being treated exhibits at least2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold,120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold,190-fold, or 200-fold increase of the abundance of one or moreBifidobacterium, Prevotella, or Desulfovibrio species after a treatmentas compared to before initiating the treatment. In another aspect, thisapplication provides a method for modulating the abundance of one ormore gut microorganisms in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising a fecal microbepreparation to achieve at least a 2-fold, 5-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold,110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold,180-fold, 190-fold, or 200-fold change of the abundance of the one ormore fecal microorganisms, wherein the one or more gut microorganismsare selected from the group consisting of Bifidobacterium, Prevotella,Desulfovibrio, Copprococcus, Clostridium, Eggerthella, and Bacteroides.In a further aspect, a treated subject exhibit a bacterial abundancefold increase (e.g., Bifidobacterium, Prevotella, or Desulfovibrio) ofbetween 2 and 10, between 3 and 10, between 4 and 10, between 5 and 10,between 6 and 10, between 10 and 20, between 10 and 30, between 10 and40, between 10 and 50, between 10 and 60, between 10 and 70, between 10and 80, between 10 and 90, between 20 and 30, between 20 and 40, between20 and 50, between 20 and 60, between 20 and 70, between 20 and 80,between 20 and 90, between 30 and 40, between 30 and 50, between 30 and60, between 30 and 70, between 30 and 80, between 30 and 90, between 40and 50, between 40 and 60, between 40 and 70, between 40 and 80, between40 and 90, between 50 and 60, between 50 and 70, between 50 and 80,between 50 and 90, between 50 and 100, between 50 and 150, between 50and 200, between 50 and 250, between 50 and 300 fold after 8 or moreweeks of treatment as compared to before initiating the treatment.

Prevotella is an anaerobic, non-spore forming, non-motile, rod thatstains Gram-negative. Desulfovibrio is a gram negative, rod shaped,sulfate reducing bacterium, which is an anaerobe and which may competewith the host for sulfur. Its metal corroding ability has led tonumerous health and safety concerns; and its production of H₂S andendotoxin can lead to genotoxic and other toxic-related problems.Bifidobacterium is an Gram-positive, non-acid-fast, non-sporeforming,and nonmotile anaerobe. Eggerthella are Gram-positive obligatelyanaerobic bacilli, which are nonmotile and do not produce endospores.Prevotella and Bifidobacterium are generally believed to be beneficialintestinal bacteria, but the role of Desulfovibrio is less understood.This application illustrates that increases in all three are beneficialin children with ASD.

In one aspect, an increase in Prevotella abundance is an increase of oneor more, two or more, three or more, four or more, five or more, six ormore, or seven or more species selected from the group consisting of P.intermedia, P. pallens, P. nigrescens, P. disiens, P. corporis, P.denticola, P. multiformis, P. melaninogenica, P. veroralis, P. oulorum,P. salivae, P. copri, P. albensis, P. bivia, P. shahii, P. loescheii, P.marshii, P. oralia, P. enoeca, P. stercorea, P. massiliensis, P.timonensis, P. buccalis, P. bergensis, P. dentalis, P. buccae, P.baroniae, P. multisaccharivorax, P. brevis, P. ruminicola, P. bryantiii,P. tannerae, P. heparinolytica, and P. zoogleoformans. In one aspect, anincrease in Prevotella abundance is an increase of one or more, two ormore, three or more, four or more, five or more, six or more, or sevenor more species listed in FIG. 20. In one aspect, an increase inDesulfovibrio abundance is an increase of one or more, two or more,three or more, four or more, five or more, six or more, or seven or moreDesulfovibrio species listed in FIG. 21. In one aspect, an increase inBifidobacterium abundance is an increase of one or more, two or more,three or more, four or more, five or more, six or more, or seven or moreBifidobacterium species selected from the group consisting of B.adolescentis, B. angulatum, B. animalis, B. asteroides, B. bifidum, B.bourn, B. breve, B. catenulatum, B. choerinum, B. coryneforme, B.cuniculi, B. denticolens, B. dentium, B. gallicum, B. gallinarum, B.indicum, B. inopinatum, B. infantis, B. lactis, B. longum, B. magnum, B.merycicum, B. minimum, B. pseudocatenulatum, B. pseudolongum, B.pullorum, B. ruminantium, B. saeculare, B. subtile, B. thermacidophilum,B. thermophilum, and B. tsurumiense. In one aspect, an increase inBifidobacterium abundance is an increase of one or more, two or more,three or more, four or more, five or more, six or more, or seven or moreBifidobacterium species listed in FIG. 22. In one aspect, a change inEggerthella abundance is an decrease of one or more or two or moreEggerthella species selected from the group consisting of Eggerthellalenta, Eggerthella sinensis, and Eggerthella hongkongensis.

In one aspect, a bacterial abundance change (e.g., increase ofBifidobacterium, Prevotella, or Desulfovibrio, or decrease ofBacteroides or Eggerthella) is ongoing during a treatment or sustainedafter finishing or discontinuing a treatment. In one aspect, a bacterialabundance change (e.g., increase of Bifidobacterium, Prevotella, orDesulfovibrio, or decrease of Bacteroides or Eggerthella) is assessed ata specific time point during or post treatment, e.g., about 2, 4, 6, 8,12, 18, 24, 32, 40, 48 weeks after initiating a treatment, or about 2,4, 6, 8, 12, 18, 24, 32, 40, 48 weeks after finishing or discontinuing atreatment. In another aspect, a bacterial abundance change (e.g.,increase of Bifidobacterium, Prevotella, or Desulfovibrio, or decreaseof Bacteroides or Eggerthella) is assessed about 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, or 110 weeks after initiating a treatment,or about 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110weeks after finishing or discontinuing a treatment.

In one aspect, a method further comprises determining in a subject arelative abundance of one or more gut microorganisms selected from thegroup consisting of Bifidobacterium, Prevotella, Desulfovibrio,Copprococcus, Clostridium, Eggerthella, and Bacteroides. In one aspect,a relative abundance is determined via an assay selected from the groupconsisting of qPCR, RT-qPCR, clone libraries, DGGE, T-RFLP, ARISA,microarrays, FIFH, dot-blot hybridization, and a DNA hybridizationmethod. In another aspect, a relative abundance is determined via 16SrDNA-targeted pyrosequencing. In a further aspect, a relative abundanceis determined via a DNA hybridization assay based on a 16S rDNAsequence. In one aspect, a relative abundance is determined viadetecting one or more proteins or metabolites specific to one or moregut microorganisms. In another aspect, a relative abundance isdetermined via an assay selected from the group consisting of2-Dimensional Gel Electrophoresis, 2-Diminsional Difference GelElectrophoresis (2D-DIGE), MALDI TOF-MS, (2D-) LC-ESI-MS/MS, AQUA and1TRAQ.

In one aspect, this application provides a method for modulating theabundance of Eggerthella or Bacteroides in a subject in need thereof,the method comprising treating the subject by administering atherapeutically effective amount of a pharmaceutical compositioncomprising a fecal microbe preparation, wherein the subject exhibits atleast a 20% change in the abundance of Eggerthella or Bacteroides afterthe treatment as compared to before initiating the treatment. In oneaspect, a subject being treated exhibits at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, or 90% change in Eggerthella or Bacteroidesabundance after a treatment as compared to before initiating thetreatment. In one aspect, a subject being treated exhibits between 20%and 30%, between 20% and 40%, between 20% and 50%, between 20% and 60%,between 20% and 70%, between 20% and 80%, between 20% and 90%, between30% and 40%, between 30% and 50%, between 30% and 60%, between 30% and70%, between 30% and 80%, between 30% and 90%, between 40% and 50%,between 40% and 60%, between 40% and 70%, between 40% and 80%, between40% and 90%, between 50% and 60%, between 50% and 70%, between 50% and80%, or between 50% and 90% change in Eggerthella or Bacteroidesabundance after a treatment as compared to before initiating thetreatment. In one aspect, a change in Eggerthella or Bacteroidesabundance is an abundance increase. In one aspect, a change inEggerthella or Bacteroides abundance is an abundance decrease.

In another aspect, this application provides a method for increasing thephylogenetic diversity of fecal microbiota, fecal phage virome, or both,of a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a fecal microbe preparation, wherein the subjectexhibits at least a two-fold increase of fecal microbiota or fecal phagevirome diversity after the treatment as compared to before initiatingthe treatment.

In one aspect, a subject being treated exhibits at least 2-fold, 3-fold,4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold,70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold,140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, or 200-foldincrease of fecal microbiota or fecal phage virome diversity after atreatment as compared to before initiating the treatment. In anotheraspect, a treated subject exhibit an increase in fecal microbiota orfecal phage virome diversity of between 2 and 10, between 3 and 10,between 4 and 10, between 5 and 10, between 6 and 10, between 10 and 20,between 10 and 30, between 10 and 40, between 10 and 50, between 10 and60, between 10 and 70, between 10 and 80, between 10 and 90, between 20and 30, between 20 and 40, between 20 and 50, between 20 and 60, between20 and 70, between 20 and 80, between 20 and 90, between 30 and 40,between 30 and 50, between 30 and 60, between 30 and 70, between 30 and80, between 30 and 90, between 40 and 50, between 40 and 60, between 40and 70, between 40 and 80, between 40 and 90, between 50 and 60, between50 and 70, between 50 and 80, between 50 and 90, between 50 and 100,between 50 and 150, between 50 and 200, between 50 and 250, between 50and 300 fold after 8 or more weeks of treatment as compared to beforeinitiating the treatment. In one aspect, an increase in fecal microbiotaor fecal phage virome diversity is ongoing during a treatment orsustained after finishing or discontinuing a treatment. In one aspect, asubject being treated exhibits at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% increase of fecal microbiota or fecal phage viromediversity after a treatment as compared to before initiating thetreatment. In another aspect, a treated subject exhibit an increase infecal microbiota or fecal phage virome diversity of between 20% and 30%,between 20% and 40%, between 20% and 50%, between 20% and 60%, between20% and 70%, between 20% and 80%, between 20% and 90%, between 30% and40%, between 30% and 50%, between 30% and 60%, between 30% and 70%,between 30% and 80%, between 30% and 90%, between 40% and 50%, between40% and 60%, between 40% and 70%, between 40% and 80%, between 40% and90%, between 50% and 60%, between 50% and 70%, between 50% and 80%, orbetween 50% and 90% after 8 or more weeks of treatment as compared tobefore initiating the treatment. In one aspect, an increase in fecalmicrobiota or fecal phage virome diversity is ongoing during a treatmentor sustained after finishing or discontinuing a treatment. In anotheraspect, an increase in fecal microbiota or fecal phage virome diversityis assessed at a specific time point during or post treatment, e.g.,about 2, 4, 6, 8, 12, 18, 24, 32, 40, 48 weeks after initiating atreatment, or about 2, 4, 6, 8, 12, 18, 24, 32, 40, 48 weeks afterfinishing or discontinuing a treatment. In another aspect, an increasein fecal microbiota or fecal phage virome diversity is assessed at 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 weeks afterinitiating a treatment, or about 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, or 110 weeks after finishing or discontinuing atreatment.

In one aspect, fecal microbiota or fecal phage virome diversity isassessed using Shannon's Index. In another aspect, fecal microbiota orfecal phage virome diversity is assessed using Faith phylogeneticdiversity. In another aspect, fecal microbiota or fecal phage viromediversity is assessed using an observed OTUs metric. In one aspect,fecal microbiota or fecal phage virome diversity is assessed using aqualitative non-phylogenetic metric (Jaccard distance), a quantitativenon-phylogenetic diversity metric (Bray-Curtis distance), a qualitativephylogenetic diversity metric (unweighted UniFrac), or a quantitativephylogenetic diversity metric (weighted UniFrac).

In one aspect, this application provides a method comprising:determining the relative abundance of one or more gut microorganisms ina subject having an ASD or having an ASD sibling, and administering atherapeutically effective amount of a pharmaceutical compositioncomprising a fecal microbe preparation to achieve at least a 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold,130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, or200-fold change of the abundance of the one or more fecalmicroorganisms, wherein the one or more gut microorganisms are from agenus selected from the group consisting of Clostridium, Bacteroides,Eggerthella, Bifidobacterium, Prevotella, and Desulfovibrio. In anotheraspect, a subject exhibits an abundance change of one or more gutmicroorganisms selected from the group consisting of Clostridium,Bacteroides, Eggerthella, Bifidobacterium, Prevotella, andDesulfovibrio, of between 3 and 10, between 4 and 10, between 5 and 10,between 6 and 10, between 10 and 20, between 10 and 30, between 10 and40, between 10 and 50, between 10 and 60, between 10 and 70, between 10and 80, between 10 and 90, between 20 and 30, between 20 and 40, between20 and 50, between 20 and 60, between 20 and 70, between 20 and 80,between 20 and 90, between 30 and 40, between 30 and 50, between 30 and60, between 30 and 70, between 30 and 80, between 30 and 90, between 40and 50, between 40 and 60, between 40 and 70, between 40 and 80, between40 and 90, between 50 and 60, between 50 and 70, between 50 and 80,between 50 and 90, between 50 and 100, between 50 and 150, between 50and 200, between 50 and 250, between 50 and 300 fold after 8 or moreweeks. In one aspect, a subject having an ASD exhibits nogastrointestinal symptom. In another aspect, a subject having an ASDdoes not exhibit one or more, two or more, three or more, four or moregastrointestinal symptoms selected from the group consisting ofabdominal pain, reflux, indigestion, irritable bowel syndrome, chronicpersistent diarrhoea, diarrhoea, flatulence, constipation, andalternating constipation/diarrhoea. In a further aspect, a subjecthaving an ASD exhibits no gastrointestinal symptom selected from thegroup consisting of abdominal pain, reflux, indigestion, irritable bowelsyndrome, chronic persistent diarrhoea, diarrhoea, flatulence,constipation, and alternating constipation/diarrhoea. In one aspect, oneor more gut microorganisms with abundance change are from Clostridium,and wherein the Clostridium microorganisms increase their abundance byat least 2 fold. In another aspect, one or more gut microorganisms withabundance change are from Bacteroides, and wherein the Bacteroidesmicroorganisms decrease their abundance by at least 2 fold. In anotheraspect, one or more gut microorganisms with abundance change are fromEggerthella, and wherein the Eggerthella microorganisms decrease theirabundance by at least 2 fold. In another aspect, one or more gutmicroorganisms with abundance change are from Bifidobacterium, andwherein the Bifidobacterium microorganisms increase their abundance byat least 2 fold. In another aspect, one or more gut microorganisms withabundance change are from Prevotella, and wherein the Prevotellamicroorganisms increase their abundance by at least 2 fold. In anotheraspect, one or more gut microorganisms with abundance change are fromDesulfovibrio, and wherein the Desulfovibrio microorganisms increasetheir abundance by at least 2 fold.

In one aspect, a subject being treated has an ASD and the treatmentimproves one or more ASD symptoms. In one aspect, a subject exhibits atleast a 10% reduction in ASD symptom severity after the treatment ascompared to before initiating the treatment, and based on an assessmentsystem selected from the group consisting of Childhood Autism RatingScale (CARS), Childhood Autism Rating Scale 2—Standard Form (CARS2-ST),Childhood Autism Rating Scale 2—High Functioning (CARS2-HF), AberrantBehavior Checklist (ABC), Social Responsiveness Scale (SRS), andVineland Adaptive Behavior Scale II (VABS-II).

In one aspect, a treatment results in an improvement of at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% based on the LeiterInternational Performance Scale (see Roid, G. H., & Miller, L. J.(1997). Leiter International Performance Scale—Revised. Wood Dale, Ill.:Stoelting) in an ASD patient. In another aspect, a Leiter scoreimprovement is measured after at least 8, 16, 24, 32, 40, 50, 60, or 80weeks of treatment and compared to a Leiter score prior to thetreatment.

One of ordinary skill in the art understands that the foregoingassessment systems are only exemplary tools for evaluating ASD-relatedsocial and cognitive symptoms. Other similar tools can be used ordesigned to evaluate core ASD-related symptoms. For example, in oneaspect, a treatment results in an improvement of at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% based on Autism Treatment EvaluationChecklist (ATEC). See Rimland and Edelson: Autism Treatment EvaluationChecklist: Statistical Analyses. Autism Research Institute 2000. Inanother aspect, a treatment results in an improvement of at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% based on PervasiveDevelopmental Disorders Behavior Inventory (PDD-BI). See Cohen et al.,The PDD Behavior Inventory: a rating scale for assessing response tointervention in children with pervasive developmental disorder. J AutismDev Disord. 2003 33(1):31-45. In yet another aspect, a treatment resultsin an improvement of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or90% based on Severity of Autism Scale (SAS). See Adams et al., Theseverity of autism is associated with toxic metal body burden and redblood cell glutathione levels. J Toxicol. 2009, 2009:532640. In afurther aspect, an improvement of autism-related symptoms or an symptomseverity reduction is assessed based on any one of the system or scalementioned in Aman et al., Outcome Measures for Clinical Drug Trials inAutism, CNS Spectr. 9(1): 36-47 (2004). In a further aspect, animprovement of autism-related symptoms or an symptom severity reductionis assessed based on any one of the symptom characterization systemslisted in Table 1. In another aspect, a method described here achieve animprovement of autism-related symptoms or an symptom severity reductionof at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 graduations in a scaledescribed here (e.g., in Table 1). In another aspect, a method describedhere achieve an improvement of autism-related symptoms or an symptomseverity reduction of between 1 and 10, between 2 and 10, between 3 and10, between 4 and 10, between 5 and 10, between 6 and 10, between 1 and9, between 2 and 9, between 3 and 9, between 4 and 9, between 5 and 9,between 6 and 9, between 1 and 8, between 2 and 8, between 3 and 8,between 4 and 8, between 5 and 8, between 6 and 8, between 1 and 7,between 2 and 7, between 3 and 7, between 4 and 7, between 5 and 7,between 6 and 7, between 1 and 6, between 2 and 6, between 3 and 6,between 4 and 6, between 5 and 6, between 1 and 5, between 2 and 5,between 3 and 5, between 4 and 5, between 1 and 4, between 2 and 4,between 3 and 4, between 1 and 3, between 2 and 3, or between 1 and 2graduations in a scale described here (e.g., CARS, CARS2-ST, CARS2-HF,ABC, SRS, VABS-II, PGI-R2, or a scale in Table 1). In one aspect, ansymptom improvement over any one of the foregoing systems is measuredafter at least 8, 16, 24, 32, 40, 50, 60, or 80 weeks of treatment andcompared to a Leiter score prior to the treatment. In one aspect, ansymptom improvement over any one of the foregoing systems is measuredafter discontinuing treatment for at least 2, 4, 6, 8, 10 or more weeksand compared to a measurement prior to the treatment.

TABLE 1 Selected outcome measures that can be used to monitor coreASD-related social and cognitive symptoms. Validated Outcome MeasuresTool Description Rater Autism Symptoms ADOS The Autism DiagnosticObservation Schedule Trained (ADOS) is a gold standards instrument forExaminer diagnosing ASD with the largest evidence base and highestsensitivity and specificity OACIS The Ohio Autism Clinical ImpressionScale was Clinician developed to be sensitive to subtle, but clinically-meaningful changes in core and associated ASD symptoms using a focusedscaling system that assesses severity and improvement in ASD behaviorssimilar to the widely used Clinical Global Impression Scale. SRS TheSocial Responsiveness Scale is a standardized Parent or and validatedquantitative scale that measures the Teacher severity and type of socialimpairments that are characteristic of ASD SCQ Social CommunicationQuestionnaire is brief Parent or instrument that evaluates communicationskills and Teacher social functioning. Both the current and lifetimeeditions will be used as appropriate AIM The Autism Impact Measure is arecently Parent developed parent-report measure that assesses bothfrequency and impact of current core ASD symptoms during the past2-weeks. Initial studies have demonstrated excellent psychometricproperties and construct validity Behavior ABC The Aberrant BehaviorChecklist is a validated Parent or questionnaire that rates symptoms ofhyperactivity, Teacher irritability, lethargy, and stereotypic behaviorin individuals with developmental disabilities. It has been used inmultiple clinical trials in ASD and has convergent and divergentvalidity CBCL Child Behavior Checklist is an easy to complete Parent orstandardized questionnaire that assesses a wide Teacher range ofbehaviors associated with ASD symptoms, including anxiety, depression,withdraw, sleep problems, somatic problems, and aggressive anddestructive behavior BASC The Behavioral Assessment System for ChildrenParent or provides scales of cognition function, behavior, Teachersocial function, and academic problems. This scale measures a wide rangeof behaviors including hyperactivity, attention, depression, anxiety,and executive function. Language CELF The Clinical Evaluations ofLanguage Trained Fundamentals is one of the only standardized, well-Examiner validated language assessment instruments that spans the agerange of most participants (using both CELF-preschool-2 and CELF-4). Itassesses a wide range of language skills that are only partiallymeasured by other language tests, including high- level language skillsthat are abnormal in individuals with ASD, such as language pragmaticsand has been used in several recent studies focusing on core languagedeficits in ASD PLS The Preschool Language Scale-4 is used in Trainedconjunction with the CELF since it is also a Examiner standardized,well-validated language assessment instrument and can measure subtlechanges in language in children with poor language abilities AdaptiveBehavior VABS The Vineland Adaptive Behavior Scale is a widely Trainedused standardized, well-validated assessment tool Interviewer forchildren with developmental delays that measures functional abilitieswithin several domains. It is particularly useful for children withintellectual disability which commonly co-occurs with ASD and has validmeasures of social impairments in children with ASD Intellect Leiter-RThe Leiter-R, due to its non-verbal nature, is an Trained excellentunbiased measure of intellect when Examiner language impairment exists.It assesses a wide range of ages (2-21 years) and contains attention andmemory batteries which are skills often disrupted in ASD. The Leiter-Ris designed to measure growth in all domains it assesses, making itsensitive to change due to treatment. Studies have shown goodpsychometric properties and verified that it is generally recommendedfor use in children with ASD WISC/ The Wechsler Intelligence Scale forChildren is one Trained WPPSI of the oldest and most widely used testsof Examiner intelligence for children. For children younger than 6 yearsthe Wechsler Preschool and Primary Scale of Intelligence test is used.One disadvantage when using this with children with ASD is its relianceon language.

In one aspect, a treatment described here achieves at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in ASD symptom severityafter 2 or more weeks of treatment as compared to before initiating thetreatment, where the ASD symptom severity is assessed by a methodselected from the group consisting of CARS, CARS2-ST, CARS2-HF, ABC,SRS, and VABS-II. In one aspect, a treatment achieves at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in ASD symptom severityafter 4 or more weeks of treatment as compared to before initiating thetreatment, where the ASD symptom severity is assessed by a methodselected from the group consisting of CARS, CARS2-ST, CARS2-HF, ABC,SRS, and VABS-II. In one aspect, a treatment achieves at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in ASD symptom severityafter 6 or more weeks of treatment as compared to before initiating thetreatment, where the ASD symptom severity is assessed by a methodselected from the group consisting of CARS, CARS2-ST, CARS2-HF, ABC,SRS, and VABS-II. In one aspect, a treatment achieves at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in ASD symptom severityafter 8 or more weeks of treatment as compared to before initiating thetreatment, where the ASD symptom severity is assessed by a methodselected from the group consisting of CARS, CARS2-ST, CARS2-HF, ABC,SRS, and VABS-II.

In another aspect, a treatment achieves between 10% and 20%, between 10%and 30%, between 10% and 40%, between 10% and 50%, between 10% and 60%,between 10% and 70%, between 10% and 80%, between 10% and 90%, between20% and 30%, between 20% and 40%, between 20% and 50%, between 20% and60%, between 20% and 70%, between 20% and 80%, between 20% and 90%,between 30% and 40%, between 30% and 50%, between 30% and 60%, between30% and 70%, between 30% and 80%, between 30% and 90%, between 40% and50%, between 40% and 60%, between 40% and 70%, between 40% and 80%,between 40% and 90%, between 50% and 60%, between 50% and 70%, between50% and 80%, or between 50% and 90% reduction in ASD symptom severityafter 8 or more weeks of treatment as compared to before initiating thetreatment, where the ASD symptom severity is assessed by a methodselected from the group consisting of CARS, CARS2-ST, CARS2-HF, ABC,SRS, and VABS-II. In another aspect, a treatment achieves between 10%and 90%, between 20% and 80%, between 30% and 70%, or between 40% and60% reduction in ASD symptom severity after 8 or more weeks of treatmentas compared to before initiating the treatment, where the ASD symptomseverity is assessed by a method selected from the group consisting ofCARS, CARS2-ST, CARS2-HF, ABC, SRS, and VABS-II. In another aspect, atreatment achieves between 10% and 90%, between 20% and 80%, between 30%and 70%, or between 40% and 60% reduction in ASD symptom severity after12 or more weeks of treatment as compared to before initiating thetreatment, where the ASD symptom severity is assessed by a methodselected from the group consisting of CARS, CARS2-ST, CARS2-HF, ABC,SRS, and VABS-II. In another aspect, a treatment achieves between 10%and 90%, between 20% and 80%, between 30% and 70%, or between 40% and60% reduction in ASD symptom severity after 18 or more weeks oftreatment as compared to before initiating the treatment, where the ASDsymptom severity is assessed by a method selected from the groupconsisting of CARS, CARS2-ST, CARS2-HF, ABC, SRS, and VABS-II. Inanother aspect, a treatment achieves between 10% and 90%, between 20%and 80%, between 30% and 70%, or between 40% and 60% reduction in ASDsymptom severity after 24 or more weeks of treatment as compared tobefore initiating the treatment, where the ASD symptom severity isassessed by a method selected from the group consisting of CARS,CARS2-ST, CARS2-HF, ABC, SRS, and VABS-II.

In one aspect, a treatment achieves at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90% reduction in ASD symptom severity andsubstantially maintains the symptom severity reduction for at least 8,12, 16, 20, 24, or 28 weeks after discontinuing the treatment, where theASD symptom severity is assessed by CARS. In one aspect, a treatmentachieves at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%reduction in ASD symptom severity and substantially maintains thesymptom severity reduction for at least 8, 12, 16, 20, 24, or 28 weeksafter discontinuing the treatment, where the ASD symptom severity isassessed by CARS2-ST. In one aspect, a treatment achieves at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in ASD symptomseverity and substantially maintains the symptom severity reduction forat least 8, 12, 16, 20, 24, or 28 weeks after discontinuing thetreatment, where the ASD symptom severity is assessed by CARS2-HF. Inone aspect, a treatment achieves at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% reduction in ASD symptom severity and substantiallymaintains the symptom severity reduction for at least 8, 12, 16, 20, 24,or 28 weeks after discontinuing the treatment, where the ASD symptomseverity is assessed by ABC. In one aspect, a treatment achieves atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in ASDsymptom severity and substantially maintains the symptom severityreduction for at least 8, 12, 16, 20, 24, or 28 weeks afterdiscontinuing the treatment, where the ASD symptom severity is assessedby SRS. In one aspect, a treatment achieves at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, or 90% reduction in ASD symptom severity andsubstantially maintains the symptom severity reduction for at least 8,12, 16, 20, 24, or 28 weeks after discontinuing the treatment, where theASD symptom severity is assessed by VABS-II.

In one aspect, a treatment achieves at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, or 90% reduction in ASD symptom severity andsubstantially maintains the symptom severity reduction for at least 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 weeks afterdiscontinuing the treatment, where the ASD symptom severity is assessedby CARS. In one aspect, a treatment achieves at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% reduction in ASD symptom severity andsubstantially maintains the symptom severity reduction for at least 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 weeks afterdiscontinuing the treatment, where the ASD symptom severity is assessedby CARS2-ST. In one aspect, a treatment achieves at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% reduction in ASD symptom severity andsubstantially maintains the symptom severity reduction for at least 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 weeks afterdiscontinuing the treatment, where the ASD symptom severity is assessedby CARS2-HF. In one aspect, a treatment achieves at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% reduction in ASD symptom severity andsubstantially maintains the symptom severity reduction for at least 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 weeks afterdiscontinuing the treatment, where the ASD symptom severity is assessedby ABC. In one aspect, a treatment achieves at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, or 90% reduction in ASD symptom severity andsubstantially maintains the symptom severity reduction for at least 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 weeks afterdiscontinuing the treatment, where the ASD symptom severity is assessedby SRS. In one aspect, a treatment achieves at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, or 90% reduction in ASD symptom severity andsubstantially maintains the symptom severity reduction for at least 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 weeks afterdiscontinuing the treatment, where the ASD symptom severity is assessedby VABS-II.

In one aspect, an ASD subject being treated exhibits no gastrointestinal(GI) symptom prior to initiating a treatment. In another aspect, an ASDsubject being treated exhibits one or more GI symptoms prior toinitiating a treatment. In one aspect, an ASD subject being treatedexhibits at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reductionin GI symptom severity after a treatment as compared to beforeinitiating the treatment. In one aspect, GI symptom severity is assessedby the Gastrointestinal Symptom Rating Scale (GSRS). In another aspect,a treatment achieves between 20% and 30%, between 20% and 40%, between20% and 50%, between 20% and 60%, between 20% and 70%, between 20% and80%, between 20% and 90%, between 30% and 40%, between 30% and 50%,between 30% and 60%, between 30% and 70%, between 30% and 80%, between30% and 90%, between 40% and 50%, between 40% and 60%, between 40% and70%, between 40% and 80%, between 40% and 90%, between 50% and 60%,between 50% and 70%, between 50% and 80%, or between 50% and 90%reduction in GI symptom severity in an ASD patient after 8 or more weeksof treatment as compared to before initiating the treatment, where theGI symptom severity is assessed by one or more symptom assessmentsystems selected from the group consisting of Gastrointestinal SeverityIndex (GSI) (Schneider et al., J Autism Dev Disord 2006; 36: 1053-64);6-item Gastrointestinal Severity Index (6-GSI) (Adams et al., BMCGastroenterology 2011, 11:22); Gastrointestinal SymptomQuestionnaire—GISQ (Bovenschen et al., Dig Dis Sci (2006) 51:1509-15);GSRS (original) (Svedlund et al., Dig Dis Sci 1988; 33: 129-134); GSRS(Likert version) (Revicki et al., Quality of Life Research. Vol 7. 199875-83); GSRS-IBS (Wiklund et al., Scand J Gastroenterol 2003; 38(9):947-954); PROMIS (Spiegel et al., Am J Gastroenterol 2014; 109(11):1804-1814); PedsQL GI Distress Domain (Varni et al., JPGN 2014; 59:347-355); Pediatric Functional Gastrointestinal Disorders (PFGD) (Caplanet al., J Pediatric Gastroenterol Nutrition 41, 305-316); Autism NetworkGI Symptom Inventory (Mazefsky, Autism Treatment Network. AutismTreatment Network GI Symptom Inventory Questionnaire, version 3.0. NewYork, N.Y.: Autism Speaks; 2005); Questionnaire on Pediatric GI Symptoms(QPGS) (ROME III criteria; Caplan et al., J Pediatric GastroenterolNutrition 2005 4, 296-304); GI Symptom Questionnaire (Chandler et al., JAutism Dev Disord 2013; 43: 2737-2747); Birmingham IBS (Roalfe et al.,BMD Gastroenterology 2008; 8:30); Gastrointestinal Symptom Score in Kids(GISSK) (Brunner et al., J Clin Rheumatol 2005; 11: 194-204); Gastro-Q(Liebbrand et al., Int J Behav Med 2002; 9: 155-72); IBS-SSS (Francis etal., Aliment Pharmacol Ther 1997; 11: 395-402); and any other similar,corresponding, or modified systems.

The GSRS is a disease-specific instrument of 15 items combined into fivesymptom clusters depicting Reflux, Abdominal pain, Indigestion,Diarrhoea and Constipation. See Svedlund et al., Dig. Dis. Sci.,33(2):129-34(1988). The GSRS has a seven-point graded Likert-type scalewhere 0 represents absence of troublesome symptoms and 3 represents anextreme degree of the symptoms with half-steps to increase thesensitivity of the scales. In one aspect, a treatment method providedhere reduces, alleviates, or eliminates one or more, two or more, threeor more, four or more, five or more, six or more, or seven or more GIsymptoms selected from the group consisting of epigastric pain, colickyabdominal pain, dull abdominal pain, undefined abdominal pain,heartburn, acid regurgitation, sucking sensations in the epigastrium,nausea and vomiting, borborygmus, abdominal distension, eructation,increased flatus, decreased passage of stools, increased passage ofstools, loose stool, hard stools, urgent need for defecation, feeling ofincomplete evacuation.

In one aspect, a treated subject's abdominal pain decreases from a moresevere level to a less severe level, where the pain levels are selectedfrom the group consisting of severe or crippling pains with impact onall social activities, prolonged and troublesome aches and pains causingrequests for relief and interfering with many social activities,occasional aches and pains interfering with some social activities, andno or transient pain.

In another aspect, a treated subject's heartburn decreases from a moresevere level to a less severe level, where the pain levels are selectedfrom the group consisting of continuous discomfort with only transientrelief by antacids, frequent episodes of prolonged discomfort; requestsfor relief, occasional discomfort of short duration, and no or transientheartburn.

In another aspect, a treated subject's acid regurgitation conditionimproves from a more severe level to a less severe level, where thecondition levels are selected from the group consisting of regurgitationseveral times a day; only transient and insignificant relief byantacids, regurgitation once or twice a day; requests for relief,occasional troublesome regurgitation, and no or transient regurgitation.

In another aspect, a treated subject's sucking sensations in theepigastrium improves from a more severe level to a less severe level,where the condition levels are selected from the group consisting ofcontinuous discomfort; frequent requests for food or antacids betweenmeals, frequent episodes of prolonged discomfort, requests for food andantacids between meals, occasional discomfort of short duration; norequests for food or antacids between meals, and no or transient suckingsensation. As used herein, sucking sensation in the epigastriumrepresents a sucking sensation in the epigastrium with relief by food orantacids. If food or antacids are not available, the sucking sensationsprogress to ache, and pains.

In another aspect, a treated subject's nausea or vomiting conditionimproves from a more severe level to a less severe level, where thecondition levels are selected from the group consisting of continuousnausea coupled with frequent vomiting, frequent and prolonged nauseawith no vomiting, occasional nausea episodes of short duration, and nonausea.

In another aspect, a treated subject's borborygmus condition improvesfrom a more severe level to a less severe level, where the conditionlevels are selected from the group consisting of continuous borborygmusseverely interfering with social performance, frequent and prolongedepisodes which can be mastered by moving without impairing socialperformance, occasional troublesome borborygmus of short duration, andno or transient borborygmus.

In another aspect, a treated subject's abdominal distension conditionimproves from a more severe level to a less severe level, where thecondition levels are selected from the group consisting of continuousdiscomfort seriously interfering with social performance, frequent andprolonged episodes which can be mastered by adjusting the clothing,occasional discomfort of short duration, and no or transient distension.

In another aspect, a treated subject's eructation condition improvesfrom a more severe level to a less severe level, where the conditionlevels are selected from the group consisting of frequent episodesseriously interfering with social performance, frequent episodesinterfering with some social activities, occasional troublesomeeructation, and no or transient eructation.

In another aspect, a treated subject's increased flatus conditionimproves from a more severe level to a less severe level, where thecondition levels are selected from the group consisting of frequentepisodes seriously interfering with social performance, frequent andprolonged episodes interfering with some social activities, occasionaldiscomfort of short duration, and no increase flatus.

In another aspect, a treated subject's decreased stool frequencyimproves from a more severe level to a less severe level, where thelevels are selected from the group consisting of every seventh day orless freluently, every sixth day, every fifth day, every fourth day,every third day, every second day, and once a day.

In another aspect, a treated subject's increased stool frequencyimproves from a more severe level to a less severe level, where thelevels are selected from the group consisting of seven times a day ormore frequently, six times a day, five times a day, four times a day,three times a day, twice a day, and once a day.

In another aspect, a treated subject's loose-stool condition improvesfrom a more severe level to a less severe level, where the levels areselected from the group consisting of watery, runny, somewhat loose, andnormal consistency.

In another aspect, a treated subject's hard-stool condition improvesfrom a more severe level to a less severe level, where the levels areselected from the group consisting of hard and fragmented withoccasional diarrhoea, hard, somewhat hard, and normal consistency.

In another aspect, a treated subject's urgency for defecation improvesfrom a more severe level to a less severe level, where the levels areselected from the group consisting of inability to control defecation,frequent feelings of urgent need for defecation with sudden need for atoilet interfering with social performance, occasional feelings ofurgent need for defecation, and normal control of defecation.

In another aspect, a treated subject's feeling of incomplete evacuationimproves from a more severe level to a less severe level, where thelevels are selected from the group consisting of defecation extremelydifficult with regular feelings of incomplete evacuation, defecationdefinitely difficult with often feelings of incomplete evacuation,defecation somewhat difficult; occasional feelings of incompleteevacuation, and feeling of complete evacuation without straining.

In another aspect, a treated subject's one or more additional GIsymptoms improve from a more severe level to a less severe level, wherethese one or more additional GI symptoms are selected from the groupconsisting of unusually large amount and/or large diameter of stools,unusually foul-smelling stools, unusual color of stool (medium brown isnormal). In another aspect, a treated subject's stool also improves to aform corresponding to Type 3 or 4 of the Bristol stool scale or improvesfrom a more irregular type to a type closer of the normal stool form(e.g., from Types 1-2 or Types 5-7 to Type 3 or 4. Bristol stool scaleis a medical aid designed to classify the form of human feces into seventypes. See Lewis and Heaton, Scand J Gastroenterol. 32(9):920-24 (1997).The seven types of stool are: Type 1: Separate hard lumps, like nuts(hard to pass); Type 2: Sausage-shaped, but lumpy; Type 3: Like asausage but with cracks on its surface; Type 4: Like a sausage or snake,smooth and soft, Type 5: Soft blobs with clear cut edges (passedeasily); Type 6: Fluffy pieces with ragged edges, a mushy stool; andType 7: Watery, no solid pieces, entirely liquid.

In one aspect, a symptom severity reduction (e.g., for ASD symptoms, GIsymptoms, or both) is ongoing during a treatment or sustained afterfinishing or discontinuing a treatment. In one aspect, a symptomseverity reduction (e.g., for ASD symptoms, GI symptoms, or both) isassessed at a specific time point during or post treatment, e.g., about2, 4, 6, 8, 12, 18, 24, 32, 40, 48 weeks after initiating a treatment,or about 2, 4, 6, 8, 12, 18, 24, 32, 40, 48 weeks after finishing ordiscontinuing a treatment. In one aspect, a symptom severity reduction(e.g., for ASD symptoms, GI symptoms, or both) is assessed at a specifictime point during or post treatment, e.g., about 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, or 110 weeks after initiating a treatment,or about 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110weeks after finishing or discontinuing a treatment.

In one aspect, a method further comprises administering an antibiotic toa subject prior to administering a pharmaceutical composition comprisinga fecal microbe preparation. In another aspect, a method furthercomprises subjecting a subject to a bowel cleanse.

In another aspect, a pharmaceutical composition used herein comprises anon-selective and substantially complete fecal microbiota supplementedwith one or more viable, non-pathogenic microorganisms selected from thegroup consisting of Bifidobacterium, Prevotella, Desulfovibrio,Copprococcus, and Clostridium. In another aspect, a pharmaceuticalcomposition used herein comprises a synthetic fecal composition ofpredetermined flora. In another aspect, a pharmaceutical compositionused herein comprises a predetermined flora comprises a preparation ofviable flora in proportional content that resembles a normal healthyhuman fecal flora and comprises no antibiotic resistant populations. Inanother aspect, a pharmaceutical composition used herein is administeredas a solid dosage form selected from the group consisting of capsule,tablet, powder, and granule. In another aspect, a pharmaceuticalcomposition used herein is formulated as an acid resistant capsule.

In another aspect, provided herein is a method of treating an autismspectrum disorder in a human subject. In exemplary aspects, the methodcomprises or consists essentially of the following steps: administeringan antibiotic to a human subject; subjecting the human subject to abowel cleanse; and administering purified fecal microbiota to the humansubject, wherein an autism spectrum disorder is treated in the humansubject.

In exemplary aspects, treating ASD comprises alleviating, ameliorating,delaying the onset of, inhibiting the progression of, or reducing theseverity of one or more, two or more, three or more, four or more, fiveor more, six or more, seven or more, eight or more symptomscharacteristic of ASD. In one aspect, a treatment alleviates,ameliorates, delays the onset of, inhibits the progression of, orreduces the severity of one or more social and cognitive coreASD-related symptoms. In some aspects, the symptom(s) is selected fromthe group consisting of: (i) insistence on sameness or resistance tochange; (ii) difficulty in expressing needs; (iii) repeating words orphrases in place of normal, responsive language; (iv) laughing, crying,showing distress for reasons not apparent to others; (v) prefers to bealone or aloof manner; (vi) tantrums; (vii) difficulty in mixing withothers; (viii) may not want to cuddle or be cuddled; (ix) little or noeye contact; (x) unresponsive to normal teaching methods; (xi) sustainedodd play; (xii) apparent over-sensitivity or under-sensitivity to pain;(xiii) little or no real fears of danger; (xiv) noticeable physicalover-activity or extreme under-activity; (xv) uneven gross/fine motorskills; and/or (xvi) non-responsiveness to verbal cues. In some aspects,the symptom(s) is selected from the group consisting of compulsivebehavior, ritualistic behavior, restricted behavior, stereotypy,sameness, or self-injury. The methods described here can lead toimprovement of any combination of the foregoing symptoms.

In exemplary aspects, the human subject exhibits a significant reductionin autism symptom severity as assessed according to a ASD rating scale.In some cases, for example, the human subject exhibits at least a 10% or20% reduction in autism symptom severity as assessed by the ChildhoodAutism Rating Scale (CARS) relative to severity as assessed prior toinitiating the method.

Subjects appropriate for treatment according to a method provided hereinmay not present with or report gastrointestinal distress symptoms priorto initiating a method as provided herein. In some cases, for example, ahuman subject appropriate for treatment according to a method providedherein manifests no gastrointestinal symptoms prior to or at the time atwhich treatment is begun. In one aspect, an ASD subject treated hereinexhibit one or more or two or more GI symptoms selected from the groupconsisting of abdominal pain, reflux, indigestion, irritable bowelsyndrome, chronic persistent diarrhoea, diarrhoea, flatulence,constipation, and alternating constipation/diarrhoea.

Regardless of the presence or absence of gastrointestinal distresssymptoms, human subjects appropriate for the methods provided hereintypically have significantly fewer species of gut bacteria before themethod of treatment as compared to a neurotypical human. In some cases,the human subject to be treated by the method exhibits at least about20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% fewer species of gut bacterialprior to administration of the purified fecal microbiota dosage ascompared to a neurotypical human.

Also provided herein are methods for reducing autism severity in anautistic human subject. In exemplary aspects, the method comprises orconsists essentially of the following steps: orally-administering anon-absorbable antibiotic to an autistic human subject; subjecting theautistic human subject to a bowel cleanse; and administering purifiedfecal microbiota from a neurotypical human donor to the human subject,wherein the human subject exhibits a significant reduction in autismsymptom severity as assessed by the Childhood Autism Rating Scale (CARS)after the method as compared to before initiating the method. In somecases, the human subject exhibits at least a 10% or 20% reduction inautism symptom severity as assessed by the Childhood Autism Rating Scale(CARS) relative to severity as assessed prior to initiating the method.

In one aspect, a fecal microbiota preparation used in a method describedhere comprises a donor's entire or substantially complete microbiota. Inone aspect, a fecal microbiota preparation comprises a non-selectivefecal microbiota. In another aspect, a fecal microbiota preparationcomprises an isolated or purified population of live non-pathogenicfecal bacteria. In a further aspect, a fecal microbiota preparationcomprises a non-selective and substantially complete fecal microbiotapreparation from a single donor. In another aspect, a therapeuticcomposition used herein comprises a mixture of live, non-pathogenic,synthetic bacteria or live, non-pathogenic, purified or extracted, fecalmicrobiota.

In one aspect, the preparation of a fecal microbiota preparationinvolves a treatment selected from the group consisting of ethanoltreatment, detergent treatment, heat treatment, irradiation, andsonication, or a combination thereof. In one aspect, the preparation ofa fecal microbiota preparation involves no treatment selected from thegroup consisting of ethanol treatment, detergent treatment, heattreatment, irradiation, and sonication. In one aspect, the preparationof a fecal microbiota preparation involves a separation step selectedfrom the group consisting of filtering, sieving, density gradients,filtration, chromatography, and a combination thereof. In one aspect,the preparation of a fecal microbiota preparation does not require oneor more separation steps selected from the group consisting offiltering, sieving, density gradients, filtration, and chromatography.In one aspect, a fecal microbiota preparation is substantially free ofnon-living matter. In one aspect, a fecal microbiota preparation issubstantially free of acellular material selected from the groupconsisting of residual fiber, DNA, viral coat material, and non-viablematerial. In one aspect, a fecal microbiota preparation is substantiallyfree of eukaryotic cells from the fecal microbiota's donor.

In one aspect, the present disclosure provides a method for treating ASDin a subject in need thereof, where the method comprises administeringto the subject a pharmaceutically active dose of a therapeuticcomposition described herein. In one aspect, the present disclosureprovides a method for treating ASD in a subject in need thereof, wherethe method comprises administering daily to the subject apharmaceutically active dose of a therapeutic composition describedherein. In one aspect, a therapeutic composition is administered to apatient in need thereof at least once daily for at least two consecutivedays. In one aspect, a therapeutic composition is administered at leastonce daily for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15consecutive days. In another aspect, a therapeutic composition isadministered at least once daily for at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 consecutive weeks. In one aspect, a therapeuticcomposition is administered at least once daily for at most 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive days orweeks. In another aspect, a therapeutic composition is administered atleast once daily for at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12consecutive weeks or months. In a further aspect, a therapeuticcomposition is administered at least once for at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 consecutive months or years, chronically for asubject's entire life span, or an indefinite period of time.

In one aspect, a therapeutic composition is administered to a patient inneed thereof at least twice daily for at least two consecutive days. Inone aspect, a therapeutic composition is administered at least twicedaily for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15consecutive days. In another aspect, a therapeutic composition isadministered at least twice daily for at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 consecutive weeks. In one aspect, a therapeuticcomposition is administered at least twice daily for at most 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive days orweek. In another aspect, a therapeutic composition is administered atleast twice daily for at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12consecutive weeks or months. In a further aspect, a therapeuticcomposition is administered at least twice for at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12 consecutive months or years, chronically for asubject's entire life span, or an indefinite period of time.

In one aspect, a therapeutic composition is administered to a patient inneed thereof at least three times daily for at least two consecutivedays. In one aspect, a therapeutic composition is administered at leastthree times daily for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,or 15 consecutive days. In another aspect, a therapeutic composition isadministered at least three times daily for at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 consecutive weeks. In one aspect, a therapeuticcomposition is administered at least three times daily for at most 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutivedays or weeks. In another aspect, a therapeutic composition isadministered at least three times daily for at most 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 consecutive weeks or months. In a further aspect, atherapeutic composition is administered at least three times for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive months oryears, chronically for a subject's entire life span, or an indefiniteperiod of time.

In one aspect, the present disclosure provides a method for treating ASDin a subject in need thereof, where the method comprises administeringorally to the subject a pharmaceutically active dose of a therapeuticcomposition comprising live, non-pathogenic, synthetic bacterial mixtureor live, non-pathogenic, purified or extracted, fecal microbiota, wherethe dose is administered at a dosing schedule of at least once or twicedaily for at least three consecutive days or weeks. In another aspect, adose is administered at least once, twice, or three times daily for aperiod between 1 and 12 weeks, between 2 and 12 weeks, between 3 and 12weeks, between 4 and 12 weeks, between 5 and 12 weeks, between 6 and 12weeks, between 7 and 12 weeks, between 8 and 12 weeks, between 9 and 12weeks, between 10 and 12 weeks, between 1 and 2 weeks, between 2 and 3weeks, between 3 and 4 weeks, between 4 and 5 weeks, between 5 and 6weeks, between 6 and 7 weeks, between 7 and 8 weeks, between 8 and 9weeks, between 9 and 10 weeks, or between 10 and 11 weeks.

In one aspect, the present disclosure provides a method for treating ASDin a subject in need thereof by administering a pharmaceuticalcomposition described herein, where the method comprises a first dosingschedule followed by a second dosing schedule. In one aspect, a firstdosing schedule comprises a treatment or induction dose. In one aspect,a first dosing schedule comprises a continuous dosing schedule. Inanother aspect, a second dosing schedule comprises a maintenance doselower than or equal to a pharmaceutically active dose of a first dosingschedule. In another aspect, a second dosing schedule lasts for at leastabout 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, 72, or 96 months. In oneaspect, a second dosing schedule lasts permanently, for a treatedsubject's entire life span, or an indefinite period of time. In oneaspect, a second dosing schedule is a continuous dosing schedule. Inanother aspect, a second dosing schedule is an intermittent dosingschedule. In a further aspect, a second dosing schedule is anintermittent dosing schedule comprising a treatment period of at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days followed by aresting period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days. In another aspect, a second dosing schedule comprisesadministering a second dose (e.g., a maintenance dose) every other day,every two days, or every 3, 4, 5, 6, 7, 8 days. In another aspect, amaintenance dose is administered for an extended period of time with orwithout titration (or otherwise changing the dosage or dosing schedule).In one aspect, the interval between a first and a second dosing scheduleis at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. Inanother aspect, a second dosing schedule (e.g., a maintenance dose)comprises a dosage about 2, 5, 10, 50, 100, 200, 400, 800, 1000, 5000 ormore folds lower than the dosage used in a first dosing schedule (e.g.,an initial treatment dose). In another aspect, a second dosing schedule(e.g., a maintenance dosing schedule) has an equal or lower dosingfrequency than a first dosing schedule (e.g., an initial treatmentdosing schedule). In another aspect, a second dosing schedule (e.g., amaintenance dosing schedule) has a higher dosing interval than a firstdosing schedule (e.g., an initial treatment dosing schedule).

In one aspect, a first or second dosing schedule used in a method can beonce-a-week, twice-a-week, or thrice-a-week. The term “once-a-week”means that a dose is administered once in a week, preferably on the sameday of each week. “Twice-a-week” means that a dose is administered twotimes in a week, preferably on the same two days of each weekly period.“Thrice-a-week” means that a dose is administered three times in a week,preferably on the same three days of each weekly period. In one aspect,a first or second dosing schedule can use fecal microbiota prepared fromtwo or more different donors. In another aspect, a first dose schedule(e.g., a treatment, induction, or initial loading dose) comprises afecal microbiota preparation from a donor different from the donorproviding the fecal microbiota preparation used in a second doseschedule (e.g., a maintenance dose).

In one aspect, a subject being treated is a subject already with adisorder (e.g., ASD). Administration of a disclosed therapeuticcomposition to clinically, asymptomatic human subject who is geneticallypredisposed or prone to a disorder (e.g., ASD) is also useful inpreventing the onset of clinical symptoms. A human subject geneticallypredisposed or prone to ASD can be a human subject having a close familymember or relative exhibiting or having suffered a disorder (e.g., ASD).In another aspect, a subject being treated is a subject in which ASD isto be prevented. In another aspect, a subject being treated ispredisposed or susceptible to a disorder (e.g., ASD). In another aspect,a subject being treated is a subject diagnosed as having a disorder(e.g., ASD). In one aspect, a subject being treated is a patient in needthereof.

In one aspect, a subject being treated is a human patient. In oneaspect, a patient is a male patient. In one aspect, a patient is afemale patient. In one aspect, a patient is a premature newborn. In oneaspect, a patient is a term newborn. In one aspect, a patient is aneonate. In one aspect, a patient is an infant. In one aspect, a patientis a toddler. In one aspect, a patient is a young child. In one aspect,a patient is a child. In one aspect, a patient is an adolescent. In oneaspect, a patient is a pediatric patient. In one aspect, a patient is ageriatric patient. In one aspect, a human patient is a child patientbelow about 18, 15, 12, 10, 8, 6, 4, 3, 2, or 1 year old. In anotheraspect, a human patient is an adult patient. In another aspect, a humanpatient is an elderly patient. In a further aspect, a human patient is apatient above about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,or 95 years old. In another aspect, a patient is about between 1 and 5,between 2 and 10, between 3 and 18, between 21 and 50, between 21 and40, between 21 and 30, between 50 and 90, between 60 and 90, between 70and 90, between 60 and 80, or between 65 and 75 years old. In oneaspect, a patient is a young old patient (65-74 years). In one aspect, apatient is a middle old patient (75-84 years). In one aspect, a patientis an old patient (>85 years).

In one aspect, a method comprises administering a therapeuticcomposition orally, by enema, or via rectal suppository. In one aspect,a pharmaceutical composition is formulated as a geltab, pill,microcapsule, capsule, or tablet. In one aspect, a therapeuticcomposition is formulated as an enteric coated capsule or microcapsule,acid-resistant capsule or microcapsule, or formulated as part of oradministered together with a food, a food additive, a dairy-basedproduct, a soy-based product or a derivative thereof, a jelly, or ayogurt. In another aspect, a therapeutic composition is formulated as anacid-resistant enteric coated capsule. A therapeutic composition can beprovided as a powder for sale in combination with a food or drink. Afood or drink can be a dairy-based product or a soy-based product. Inanother aspect, a food or food supplement contains enteric-coated and/oracid-resistant microcapsules containing a therapeutic composition.

In an aspect, a therapeutic composition comprises a liquid culture. Inanother aspect, a therapeutic composition is lyophilized, pulverized andpowdered. It may then be infused, dissolved such as in saline, as anenema. Alternatively the powder may be encapsulated as enteric-coatedand/or acid-resistant capsules for oral administration. These capsulesmay take the form of enteric-coated and/or acid-resistant microcapsules.A powder can preferably be provided in a palatable form forreconstitution for drinking or for reconstitution as a food additive. Ina further aspect, a food is yogurt. In one aspect, a powder may bereconstituted to be infused via naso-duodenal infusion.

In another aspect, a therapeutic composition is in a liquid, frozen,freeze-dried, spray-dried, lyophilized, or powder formulation. In afurther aspect, a therapeutic composition is formulated as a delayed orgradual enteric release form. In another aspect, a therapeuticcomposition comprises an excipient, a saline, a buffer, a bufferingagent, or a fluid-glucose-cellobiose agar (RGCA) media.

In one aspect, a therapeutic composition further comprises an acidsuppressant, an antacid, an H2 antagonist, a proton pump inhibitor or acombination thereof. In one aspect, a therapeutic compositionsubstantially free of non-living matter. In another aspect, atherapeutic composition substantially free of acellular materialselected from the group consisting of residual fiber, DNA, viral coatmaterial, and non-viable material.

In one aspect, a therapeutic composition comprises a cryoprotectant. Inanother aspect, a cryoprotectant comprises, consisting essentially or,or consisting of polyethylene glycol, skim milk, erythritol, arabitol,sorbitol, glucose, fructose, alanine, glycine, proline, sucrose,lactose, ribose, trehalose, dimethyl sulfoxide (DMSO), glycerol, or acombination thereof.

In another aspect, a therapeutic composition comprises a lyoprotectant.In one aspect, the same substance or the same substance combination isused as both a cryoprotectant and a lyoprotectant. Exemplarylyoprotectants include sugars such as sucrose or trehalose; an aminoacid such as monosodium glutamate or histidine; a methylamine such asbetaine; a lyotropic salt such as magnesium sulfate; a polyol such astrihydric or higher sugar alcohols, e.g. glycerin, erythritol, glycerol,arabitol, xylitol, sorbitol, and mannitol; propylene glycol;polyethylene glycol; Pluronics; and combinations thereof. In one aspect,a lyoprotectant is a non-reducing sugar, such as trehalose or sucrose.In one aspect, a cryoprotectant or a lyoprotectant consistingessentially of, or consisting of, one or more substances mentioned inthis paragraph and the paragraph above.

In one aspect, a lyophilized formulation comprises trehalose. In oneaspect, a lyophilized formulation comprises 2% to 30%, 3% to 25%, 4% to20%, 5% to 15%, 6% to 10%, 2% to 30%, 2% to 25%, 2% to 20%, 2% to 15%,or 2% to 10% trehalose. In one aspect, a lyophilized formulationcomprises at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15%trehalose. In one aspect, a lyophilized formulation comprises at most2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% trehalose. In one aspect, alyophilized formulation comprises about 5% trehalose. In one aspect, alyophilized formulation comprises trehalose and sucrose. In one aspect,a lyophilized formulation comprises between about 8% to 12% trehalosewith between about 1.5% to 3.5% sucrose and between about 0.5% to 1.5%NaCl.

In one aspect, a therapeutic composition also comprises or issupplemented with a prebiotic nutrient selected from the groupconsisting of polyols, fructooligosaccharides (FOSs), oligofructoses,inulins, galactooligosaccharides (GOSs), xylooligosaccharides (XOSs),polydextroses, monosaccharides, tagatose, and/or mannooligosaccharides.

In one aspect, a method further comprises pretreating a subject with anantibiotic composition prior to administering a therapeutic bacterial ormicrobiota composition. In one aspect, an antibiotic compositioncomprises an antibiotic selected from the group consisting of rifabutin,clarithromycin, clofazimine, vancomycin, rifampicin, nitroimidazole,chloramphenicol, and a combination thereof. In another aspect, anantibiotic composition comprises an antibiotic selected from the groupconsisting of rifaximin, rifamycin derivative, rifampicin, rifabutin,rifapentine, rifalazil, bicozamycin, aminoglycoside, gentamycin,neomycin, streptomycin, paromomycin, verdamicin, mutamicin, sisomicin,netilmicin, retymicin, kanamycin, aztreonam, aztreonam macrolide,clarithromycin, dirithromycin, roxithromycin, telithromycin,azithromycin, bismuth subsalicylate, vancomycin, streptomycin,fidaxomicin, amikacin, arbekacin, neomycin, netilmicin, paromomycin,rhodostreptomycin, tobramycin, apramycin, and a combination thereof. Ina further aspect, a method further comprises pretreating a subject withan anti-inflammatory drug prior to administration of a therapeuticbacterial or microbiota composition.

In one aspect, every about 200 mg of a pharmaceutical compositioncomprises a pharmacologically active dose. In one aspect, every about75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000,1500, or 2000 mg of a pharmaceutical composition comprises apharmacologically active dose.

In one aspect, a pharmaceutically active or therapeutic effective dosecomprises at least about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², or10¹³ cfu. In another aspect, a pharmaceutically active therapeuticeffective dose comprises at most about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰,10¹¹, 10¹², or 10¹³ cfu. In a further aspect, a pharmacologically activetherapeutic effective dose is selected from the group consisting of from10⁸ cfu to 10¹⁴ cfu, from 10⁹ cfu to 10¹³ cfu, from 10¹⁰ cfu to 10¹²cfu, from 10⁹ cfu to 10¹⁴ cfu, from 10⁹ cfu to 10¹² cfu, from 10⁹ cfu to10¹¹ cfu, from 10⁹ cfu to 10¹⁰ cfu, from 10¹⁰ cfu to 10¹⁴ cfu, from 10¹⁰cfu to 10¹³ cfu, from 10¹¹ cfu to 10¹⁴ cfu, from 10¹¹ cfu to 10¹³ cfu,from 10¹² cfu to 10¹⁴ cfu, and from 10¹³ cfu to 10¹⁴ cfu. In one aspect,a pharmaceutical composition comprises the foregoing pharmaceuticallyactive or therapeutic effective dose in a unit weight of about 0.2, 0.4,0.6, 0.8 or 1.0 gram, or a unit volume of about 0.2, 0.4, 0.6, 0.8 or1.0 milliliter.

In one aspect, a pharmaceutically active or therapeutic effective dosecomprises at least about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², or10¹³ cells or spores. In another aspect, a pharmaceutically active ortherapeutic effective dose comprises at most about 10⁵, 10⁶, 10⁷, 10⁸,10⁹, 10¹⁰, 10¹¹, 10¹², or 10¹³ total cells or spores. In a furtheraspect, a pharmacologically active or therapeutic effective dose isselected from the group consisting of from 10⁸ to 10¹⁴, from 10⁹ to10¹³, from 10¹⁰ to 10¹², from 10⁹ to 10¹⁴, from 10⁹ to 10¹², from 10⁹ to10¹¹, from 10⁹ to 10¹⁰, from 10¹⁰ to 10¹⁴, from 10¹⁰ to 10¹³, from 10¹¹to 10¹⁴, from 10¹¹ to 10¹³, from 10¹² to 10¹⁴, and from 10¹³ to 10¹⁴cells or spores. In an aspect, the pharmaceutically active ortherapeutic effective dose cell count is directed to live cells. In oneaspect, a pharmaceutical composition comprises the foregoingpharmaceutically active or therapeutic effective dose in a unit weightof about 0.2, 0.4, 0.6, 0.8 or 1.0 gram, or a unit volume of about 0.2,0.4, 0.6, 0.8 or 1.0 milliliter.

In one aspect, a therapeutic composition described and used herecomprises one or more, two or more, three or more, four or more, or fiveor more isolated, purified, or cultured microorganisms selected from thegroup consisting of Clostridium, Bacillus, Collinsella, Bacteroides,Eggerthella, Eubacterium, Fusobacterium, Propionibacterium,Lactobacillus, Ruminococcus, Escherichia coli, Gemmiger, Desulfomonas,Peptostreptococcus, Bifidobacterium, Coprococcus, Dorea, and Monilia.

In one aspect, a fecal microbiota preparation described herein comprisesa purified or reconstituted fecal bacterial mixture. In one aspect, afecal microbiota preparation described and used here comprises one ormore, one or more, two or more, three or more, four or more, or five ormore live fecal microorganisms are selected from the group consisting ofAcidaminococcus, Akkermansia, Alistipes, Anaerotruncus, Bacteroides,Eggerthella, Bifidobacterium, Blautia, Butyrivibrio, Clostridium,Collinsella, Coprococcus, Corynebacterium, Dorea, Enterococcus,Escherichia, Eubacterium, Faecalibacterium, Haemophilus, Holdemania,Lactobacillus, Moraxella, Parabacteroides, Prevotella,Propionibacterium, Raoultella, Roseburia, Ruminococcus, Staphylococcus,Streptococcus, Subdoligranulum, and Veillonella. In one aspect, a fecalmicrobiota preparation comprises one or more, one or more, two or more,three or more, four or more, or five or more live fecal microorganismsare selected from the group consisting of Bacteroides fragilis ssp.vulgatus, Collinsella aerofaciens, Bacteroides fragilis ssp.thetaiotaomicron, Peptostreptococcus productus II, Parabacteroidesdistasonis, Faecalibacterium prausnitzii, Coprococcus eutactus,Peptostreptococcus productus I, Ruminococcus bromii, Bifidobacteriumadolescentis, Gemmiger formicilis, Bifidobacterium longum, Eubacteriumsiraeum, Ruminococcus torques, Eubacterium rectale, Eubacterium eligens,Bacteroides eggerthii, Clostridium leptum, Bacteroides fragilis ssp. A,Eubacterium biforme, Bifidobacterium infantis, Eubacterium rectale,Coprococcus comes, Pseudoflavonifractor capillosus, Ruminococcus albus,Dorea formicigenerans, Eubacterium hallii, Eubacterium ventriosum I,Fusobacterium russi, Ruminococcus obeum, Eubacterium rectale,Clostridium ramosum, Lactobacillus leichmannii, Ruminococcus callidus,Butyrivibrio crossotus, Acidaminococcus fermentans, Eubacteriumventriosum, Bacteroides fragilis ssp. fragilis, Coprococcus catus,Aerostipes hadrus, Eubacterium cylindroides, Eubacterium ruminantium,Staphylococcus epidermidis, Eubacterium limosum, Tissirella praeacuta,Fusobacterium mortiferum I, Fusobacterium naviforme, Clostridiuminnocuum, Clostridium ramosum, Propionibacterium acnes, Ruminococcusflavefaciens, Bacteroides fragilis ssp. ovatus, Fusobacterium nucleatum,Fusobacterium mortiferum, Escherichia coli, Gemella morbillorum,Finegoldia magnus, Streptococcus intermedius, Ruminococcus lactaris,Eubacterium tenue, Eubacterium ramulus, Bacteroides clostridiiformisssp. clostridliformis, Bacteroides coagulans, Prevotella oralis,Prevotella ruminicola, Odoribacter splanchnicus, and Desuifomonas pigra.

In one aspect, a fecal microbiota preparation described and used herelacks or is substantially devoid of one or more, one or more, two ormore, three or more, four or more, or five or more live fecalmicroorganisms are selected from the group consisting ofAcidaminococcus, Akkermansia, Alistipes, Anaerotruncus, Bacteroides,Eggerthella, Bifidobacterium, Blautia, Butyrivibrio, Clostridium,Collinsella, Coprococcus, Corynebacterium, Dorea, Enterococcus,Escherichia, Eubacterium, Faecalibacterium, Haemophilus, Holdemania,Lactobacillus, Moraxella, Parabacteroides, Prevotella,Propionibacterium, Raoultella, Roseburia, Ruminococcus, Staphylococcus,Streptococcus, Subdoligranulum, and Veillonella. In one aspect, a fecalmicrobiota preparation lacks or is substantially devoid of one or more,one or more, two or more, three or more, four or more, or five or livemore fecal microorganisms are selected from the group consisting ofBacteroides fragilis ssp. vulgatus, Collinsella aerofaciens, Bacteroidesfragilis ssp. thetaiotaomicron, Peptostreptococcus productus II,Parabacteroides distasonis, Faecalibacterium prausnitzii, Coprococcuseutactus, Peptostreptococcus productus I, Ruminococcus bromii,Bifidobacterium adolescentis, Gemmiger formicilis, Bifidobacteriumlongum, Eubacterium siraeum, Ruminococcus torques, Eubacterium rectale,Eubacterium eligens, Bacteroides eggerthii, Clostridium leptum,Bacteroides fragilis ssp. A, Eubacterium biforme, Bifidobacteriuminfantis, Eubacterium rectale, Coprococcus comes, Pseudoflavonifractorcapillosus, Ruminococcus albus, Dorea formicigenerans, Eubacteriumhallii, Eubacterium ventriosum I, Fusobacterium russi, Ruminococcusobeum, Eubacterium rectale, Clostridium ramosum, Lactobacillusleichmannii, Ruminococcus callidus, Butyrivibrio crossotus,Acidaminococcus fermentans, Eubacterium ventriosum, Bacteroides fragilisssp. fragilis, Coprococcus catus, Aerostipes hadrus, Eubacteriumcylindroides, Eubacterium ruminantium, Staphylococcus epidermidis,Eubacterium limosum, Tissirella praeacuta, Fusobacterium mortiferum I,Fusobacterium naviforme, Clostridium innocuum, Clostridium ramosum,Propionibacterium acnes, Ruminococcus flavefaciens, Bacteroides fragilisssp. ovatus, Fusobacterium nucleatum, Fusobacterium mortiferum,Escherichia coli, Gemella morbillorum, Finegoldia magnus, Streptococcusintermedius, Ruminococcus lactaris, Eubacterium tenue, Eubacteriumramulus, Bacteroides clostridiiformis ssp. clostridliformis, Bacteroidescoagulans, Prevotella oralis, Prevotella ruminicola, Odoribactersplanchnicus, and Desuifomonas pigra.

In another aspect, a therapeutic composition comprises a fecalmicrobiota further supplemented, spiked, or enhanced with a fecalmicroorganism. In one aspect, a fecal microbiota is supplemented with anon-pathogenic (or with attenuated pathogenicity) bacterium ofClostridium, Collinsella, Dorea, Ruminococcus, Coprococcus, Prevotella,Veillonella, Bacteroides, Eggerthella, Bacillus, or a combinationthereof. In another aspect, a therapeutic composition comprises a fecalmicrobiota further supplemented, spiked, or enhanced with a species ofVeillonellaceae, Firmicutes, Gammaproteobacteria, Bacteroidetes, or acombination thereof. In another aspect, a therapeutic compositioncomprises a fecal microbiota further supplemented with fecal bacterialspores. In one aspect, fecal bacterial spores are Clostridium spores,Bacillus spores, or both. In another aspect, a therapeutic compositioncomprises a fecal microbiota further supplemented, spiked, or enhancedwith a Bacteroides species selected from the group consisting ofBacteroides coprocola, Bacteroides plebeius, Bacteroides massiliensis,Bacteroides vulgatus, Bacteroides helcogenes, Bacteroides pyogenes,Bacteroides tectus, Bacteroides uniformis, Bacteroides stercoris,Bacteroides eggerthii, Bacteroides finegoldii, Bacteroidesthetaiotaomicron, Bacteroides ovatus, Bacteroides acidifaciens,Bacteroides caccae, Bacteroides nordii, Bacteroides salyersiae,Bacteroides fragilis, Bacteroides intestinalis, Bacteroides coprosuis,Bacteroides distasonis, Bacteroides goldsteinii, Bacteroides merdae,Bacteroides forsythus, Bacteroides splanchnicus, Bacteroides capillosus,Bacteroides cellulosolvens, and Bacteroides ureolyticus.

In an aspect, a therapeutic composition comprises a fecal microbiotafrom a subject selected from the group consisting of a human, a bovine,a dairy calf, a ruminant, an ovine, a caprine, or a cervine. In anotheraspect, a therapeutic composition can be administered to a subjectselected from the group consisting of a human, a bovine, a dairy calf, aruminant, an ovine, a caprine, or a cervine. In an aspect, a therapeuticcomposition is substantially or nearly odourless.

In an aspect, a therapeutic composition provided here comprises a fecalmicrobiota preparation comprising a Shannon Diversity Index of greaterthan or equal to 0.3, greater than or equal to 0.4, greater than orequal to 0.5, greater than or equal to 0.6, greater than or equal to0.7, greater than or equal to 0.8, greater than or equal to 0.9, greaterthan or equal to 1.0, greater than or equal to 1.1, greater than orequal to 1.2, greater than or equal to 1.3, greater than or equal to1.4, greater than or equal to 1.5, greater than or equal to 1.6, greaterthan or equal to 1.7, greater than or equal to 1.8, greater than orequal to 1.9, greater than or equal to 2.0, greater than or equal to2.1, greater than or equal to 2.2, greater than or equal to 2.3, greaterthan or equal to 2.4, greater than or equal to 2.5, greater than orequal to 3.0, greater than or equal to 3.1, greater than or equal to3.2, greater than or equal to 3.3, greater than or equal to 3.4, greaterthan or equal to 3.5, greater than or equal to 3.6, greater than orequal to 3.7, greater than or equal to 3.8, greater than or equal to3.9, greater than or equal to 4.0, greater than or equal to 4.1, greaterthan or equal to 4.2, greater than or equal to 4.3, greater than orequal to 4.4, greater than or equal to 4.5, or greater than or equal to5.0. In another aspect, a therapeutic composition comprises fecalmicrobiota comprising a Shannon Diversity Index of between 0.1 and 3.0,between 0.1 and 2.5, between 0.1 and 2.4, between 0.1 and 2.3, between0.1 and 2.2, between 0.1 and 2.1, between 0.1 and 2.0, between 0.4 and2.5, between 0.4 and 3.0, between 0.5 and 5.0, between 0.7 and 5.0,between 0.9 and 5.0, between 1.1 and 5.0, between 1.3 and 5.0, between1.5 and 5.0, between 1.7 and 5.0, between 1.9 and 5.0, between 2.1 and5.0, between 2.3 and 5.0, between 2.5 and 5.0, between 2.7 and 5.0,between 2.9 and 5.0, between 3.1 and 5.0, between 3.3 and 5.0, between3.5 and 5.0, between 3.7 and 5.0, between 31.9 and 5.0, or between 4.1and 5.0. In one aspect, a Shannon Diversity Index is calculated at thephylum level. In another aspect, a Shannon Diversity Index is calculatedat the family level. In one aspect, a Shannon Diversity Index iscalculated at the genus level. In another aspect, a Shannon DiversityIndex is calculated at the species level. In a further aspect, atherapeutic composition comprises a preparation of flora in proportionalcontent that resembles a normal healthy human fecal flora.

In a further aspect, a therapeutic composition comprises fecal bacteriafrom at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different families. In anaspect, a therapeutic composition provided here comprises a fecalmicrobiota comprising a weight ratio between fecal-derived non-livingmaterial and fecal-derived biological material of no greater than 0.05%,0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, or 10%. In another aspect, a therapeutic compositionprovided here comprises a fecal microbiota comprising a weight ratiobetween fecal-derived non-living material and fecal-derived biologicalmaterial of no greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95%. In another aspect, a therapeuticcomposition provided here comprises, consists of, or consistsessentially of, particles of non-living material and/or particles ofbiological material of a fecal sample that passes through a sieve, acolumn, or a similar filtering device having a sieve, exclusion, orparticle filter size of 2.0 mm, 1.0 mm, 0.5 mm, 0.25 mm, 0.212 mm, 0.180mm, 0.150 mm, 0.125 mm, 0.106 mm, 0.090 mm, 0.075 mm, 0.063 mm, 0.053mm, 0.045 mm, 0.038 mm, 0.032 mm, 0.025 mm, 0.020 mm, 0.01 mm, or 0.2mm. “Non-living material” does not include an excipient, e.g., apharmaceutically inactive substance, such as a cryoprotectant, added toa processed fecal material. “Biological material” refers to the livingmaterial in fecal material, and includes microbes including prokaryoticcells, such as bacteria and archaea (e.g., living prokaryotic cells andspores that can sporulate to become living prokaryotic cells),eukaryotic cells such as protozoa and fungi, and viruses. In one aspect,“biological material” refers to the living material, e.g., the microbes,eukaryotic cells, and viruses, which are present in the colon of anormal healthy human. In an aspect, a therapeutic composition providedor comprises an extract of human feces where the composition issubstantially odorless. In an aspect, a therapeutic composition providedor comprises fecal material or a fecal floral preparation in alyophilized, crude, semi-purified or purified formulation.

In an aspect, a fecal microbiota in a therapeutic composition compriseshighly refined or purified fecal flora, e.g., substantially free ofnon-floral fecal material. In an aspect, a fecal microbiota can befurther processed, e.g., to undergo microfiltration before, after, orbefore and after sieving. In another aspect, a highly purified fecalmicrobiota product is ultra-filtrated to remove large molecules butretain the therapeutic microflora, e.g., bacteria.

In another aspect, a fecal microbiota in a therapeutic composition usedherein comprises or consists essentially of a substantially isolated ora purified fecal flora or entire (or substantially entire) microbiotathat is (or comprises) an isolate of fecal flora that is at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%,99.8% or 99.9% isolated or pure, or having no more than about 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% or more non-fecalfloral material; or, a substantially isolated, purified, orsubstantially entire microbiota as described in Sadowsky et al., WO2012/122478 A1, or as described in Borody et al., WO 2012/016287 A2. Inone aspect, a fecal microbiota preparation comprises a weight ratiobetween fecal-derived non-living material and fecal-derived biologicalmaterial of no greater than about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, 2%, 5%, 8%, 10%, 15%, 20%, 30%, 40$, or 50%.

In an aspect, a fecal microbiota in a therapeutic composition comprisesa donor's substantially entire or non-selective fecal microbiota,reconstituted fecal material, or synthetic fecal material. In anotheraspect, the fecal microbiota in a therapeutic composition comprises noantibiotic resistant population. In another aspect, a therapeuticcomposition comprises a fecal microbiota and is largely free ofextraneous matter (e.g., non-living matter including acellular mattersuch as residual fiber, DNA, RNA, viral coat material, non-viablematerial; and living matter such as eukaryotic cells from the fecalmatter's donor).

In an aspect, a fecal microbiota in a therapeutic composition usedherein is derived from disease-screened fresh homologous feces orequivalent freeze-dried and reconstituted feces. In an aspect, a freshhomologous feces does not include an antibiotic resistant population. Inanother aspect, a fecal microbiota in a therapeutic composition isderived from a synthetic fecal composition. In an aspect, a syntheticfecal composition comprises a preparation of viable flora whichpreferably in proportional content, resembles normal healthy human fecalflora which does not include antibiotic resistant populations. Suitablemicroorganisms may be selected from the following: Bacteroides,Eggerthella, Eubacterium, Fusobacterium, Propionibacterium,Lactobacillus, Ruminococcus, Escherichia coli, Gemmiger, Clostridium,Desulfomonas, Peptostreptococcus, Bifidobacterium, Collinsella,Coprococcus, Dorea, and Ruminococcus.

In an aspect, a therapeutic composition is combined with other adjuvantssuch as antacids to dampen bacterial inactivation in the stomach. (e.g.,Mylanta, Mucaine, Gastrogel). In another aspect, acid secretion in thestomach could also be pharmacologically suppressed using H2-antagonistsor proton pump inhibitors. An example H2-antagonist is ranitidine. Anexample proton pump inhibitor is omeprazole. In one aspect, an acidsuppressant is administered prior to administering, or inco-administration with, a therapeutic composition.

In an aspect, a therapeutic composition is administered in the form of:an enema composition which can be reconstituted with an appropriatediluent; enteric-coated capsules; enteric-coated microcapsules;acid-resistant tablet; acid-resistant capsules; acid-resistantmicrocapsules; powder for reconstitution with an appropriate diluent fornaso-enteric infusion or colonoscopic infusion; powder forreconstitution with appropriate diluent, flavoring and gastric acidsuppression agent for oral ingestion; powder for reconstitution withfood or drink; or food or food supplement comprising enteric-coatedand/or acid-resistant microcapsules of the composition, powder, jelly,or liquid.

In an aspect, a treatment method effects a cure, reduction of thesymptoms, or a percentage reduction of symptoms of a disorder (e.g.,ASD). The change of flora is preferably as “near-complete” as possibleand the flora is replaced by viable organisms which will crowd out anyremaining, original flora. Typically the change in enteric floracomprises introduction of an array of predetermined flora into thegastro-intestinal system, and thus in a preferred form the method oftreatment comprises substantially or completely displacing pathogenicenteric flora in patients requiring such treatment.

In another aspect, a therapeutic composition can be provided togetherwith a pharmaceutically acceptable carrier. As used herein, a“pharmaceutically acceptable carrier” refers to a non-toxic solvent,dispersant, excipient, adjuvant, or other material which is mixed with alive bacterium in order to permit the formation of a pharmaceuticalcomposition, e.g., a dosage form capable of administration to thepatient. A pharmaceutically acceptable carrier can be liquid (e.g.,saline), gel or solid form of diluents, adjuvant, excipients or an acidresistant encapsulated ingredient. Suitable diluents and excipientsinclude pharmaceutical grades of physiological saline, dextrose,glycerol, mannitol, lactose, starch, magnesium stearate, sodiumsaccharin, cellulose, magnesium carbonate, and the like, andcombinations thereof. In another aspect, a therapeutic composition maycontain auxiliary substances such as wetting or emulsifying agents,stabilizing or pH buffering agents. In an aspect, a therapeuticcomposition contains about 1%-5%, 5%-10%, 10%-15%, 15-20%, 20%-25%,25-30%, 30-35%, 40-45%, 50%-55%, 1%-95%, 2%-95%, 5%-95%, 10%-95%,15%-95%, 20%-95%, 25%-95%, 30%-95%, 35%-95%, 40%-95%, 45%-95%, 50%-95%,55%-95%, 60%-95%, 65%-95%, 70%-95%, 45%-95%, 80%-95%, or 85%-95% ofactive ingredient. In an aspect, a therapeutic composition containsabout 2%-70%, 5%-60%, 10%-50%, 15%-40%, 20%-30%, 25%-60%, 30%-60%, or35%-60% of active ingredient.

In an aspect, a therapeutic composition can be incorporated intotablets, drenches, boluses, capsules or premixes. Formulation of theseactive ingredients into such dosage forms can be accomplished by meansof methods well known in the pharmaceutical formulation arts. See, e.g.,U.S. Pat. No. 4,394,377. Filling gelatin capsules with any desired formof the active ingredients readily produces capsules. If desired, thesematerials can be diluted with an inert powdered diluent, such as sugar,starch, powdered milk, purified crystalline cellulose, or the like toincrease the volume for convenience of filling capsules.

In an aspect, conventional formulation processes can be used to preparetablets containing a therapeutic composition. In addition to the activeingredients, tablets may contain a base, a disintegrator, an absorbent,a binder, and a lubricant. Typical bases include lactose, sugar, sodiumchloride, starch and mannitol. Starch is also a good disintegrator as isalginic acid. Surface-active agents such as sodium lauryl sulfate anddioctyl sodium sulphosuccinate are also sometimes used. Commonly usedabsorbents include starch and lactose. Magnesium carbonate is alsouseful for oily substances. As a binder there can be used, for example,gelatin, gums, starch, dextrin, polyvinyl pyrrolidone and variouscellulose derivatives. Among the commonly used lubricants are magnesiumstearate, talc, paraffin wax, various metallic soaps, and polyethyleneglycol.

In an aspect, for preparing solid compositions such as tablets, anactive ingredient is mixed with a pharmaceutical carrier, e.g.,conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, or other pharmaceutical diluents, e.g. water, to forma solid preformulation composition containing a homogeneous mixture of acomposition of the present disclosure. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulationcomposition is then subdivided into unit dosage forms of the typedescribed above containing a desired amount of an active ingredient(e.g., at least about 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², or 10¹³cfu). A therapeutic composition used herein can be flavored.

In an aspect, a therapeutic composition can be a tablet or a pill. Inone aspect, a tablet or a pill can be coated or otherwise compounded toprovide a dosage form affording the advantage of prolonged action. Forexample, a tablet or pill can comprise an inner dosage and an outerdosage component, the latter being in the form of an envelope over theformer. The two components can be separated by an enteric layer whichserves to resist disintegration in the stomach and permits the innercomponent to pass intact into the duodenum or to be delayed in release.A variety of materials can be used for such enteric layers or coatings,such materials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol andcellulose acetate.

In an aspect, a therapeutic composition is formulated as a delayed orgradual enteric release form. In an aspect, a delayed or gradual entericrelease formulation comprises the use of cellulose acetate, polyethyleneglycerol, or both. In an aspect, a delayed or gradual enteric releaseformulation comprises the use of a hydroxypropylmethylcellulose (HPMC),a microcrystalline cellulose (MCC), magnesium stearate, or a combinationthereof. In an aspect, a delayed or gradual enteric release formulationcomprises the use of a poly(meth)acrylate, a methacrylic acid copolymerB, a methyl methacrylate, a methacrylic acid ester, apolyvinylpyrrolidone (PVP), a PVP-K90, or a combination thereof. In anaspect, a delayed or gradual enteric release formulation comprises theuse of a solid inner layer sandwiched between two outer layers; whereinthe solid inner layer comprises the pharmaceutical composition andanother component selected from the group consisting of a disintegrant,an exploding agent, an effervescent or any combination thereof; whereinthe outer layer comprises a substantially water soluble, a crystallinepolymer, or both. In an aspect, a delayed or gradual enteric releaseformulation comprises the use of a non-swellable diffusion matrix.

In another aspect, a delayed or gradual enteric release formulationcomprises the use of a bilayer tablet or capsule which comprises a firstlayer comprising a polyalkylene oxide, a polyvinylpyrrolidone, alubricant, or a mixture thereof, and a second osmotic push layercomprising polyethylene oxide, carboxy-methylcellulose, or both. In anaspect, a delayed or gradual enteric release formulation comprises theuse of a release-retarding matrix material selected from the groupconsisting of an acrylic polymer, a cellulose, a wax, a fatty acid,shellac, zein, hydrogenated vegetable oil, hydrogenated castor oil,polyvinylpyrrolidine, a vinyl acetate copolymer, a vinyl alcoholcopolymer, polyethylene oxide, an acrylic acid and methacrylic acidcopolymer, a methyl methacrylate copolymer, an ethoxyethyl methacrylatepolymer, a cyanoethyl methacrylate polymer, an aminoalkyl methacrylatecopolymer, a poly(acrylic acid), a poly(methacrylic acid), a methacrylicacid alkylamide copolymer, a poly(methyl methacrylate), apoly(methacrylic acid anhydride), a methyl methacrylate polymer, apolymethacrylate, a poly(methyl methacrylate) copolymer, apolyacrylamide, an aminoalkyl methacrylate copolymer, a glycidylmethacrylate copolymer, a methyl cellulose, an ethylcellulose, acarboxymethylcellulose, a hydroxypropylmethylcellulose, a hydroxymethylcellulose, a hydroxyethyl cellulose, a hydroxypropyl cellulose, acrosslinked sodium carboxymethylcellulose, a crosslinkedhydroxypropylcellulose, a natural wax, a synthetic wax, a fatty alcohol,a fatty acid, a fatty acid ester, a fatty acid glyceride, a hydrogenatedfat, a hydrocarbon wax, stearic acid, stearyl alcohol, beeswax,glycowax, castor wax, carnauba wax, a polylactic acid, polyglycolicacid, a co-polymer of lactic and glycolic acid, carboxymethyl starch,potassium methacrylate/divinylbenzene copolymer, crosslinkedpolyvinylpyrrolidone, poly inylalcohols, polyvinylalcohol copolymers,polyethylene glycols, non-crosslinked polyvinylpyrrolidone,polyvinylacetates, polyvinylacetate copolymers, or any combinationthereof. In an aspect, a delayed or gradual enteric release formulationcomprises the use of a microenvironment pH modifier.

In an aspect, a therapeutic composition can be a drench. In one aspect,a drench is prepared by choosing a saline-suspended form of atherapeutic composition. A water-soluble form of one ingredient can beused in conjunction with a water-insoluble form of the other bypreparing a suspension of one with an aqueous solution of the other.Water-insoluble forms of either active ingredient may be prepared as asuspension or in some physiologically acceptable solvent such aspolyethylene glycol. Suspensions of water-insoluble forms of eitheractive ingredient can be prepared in oils such as peanut, corn, sesameoil or the like; in a glycol such as propylene glycol or a polyethyleneglycol; or in water depending on the solubility of a particular activeingredient. Suitable physiologically acceptable adjuvants may benecessary in order to keep the active ingredients suspended. Adjuvantscan include and be chosen from among the thickeners, such ascarboxymethylcellulose, polyvinyl pyrrolidone, gelatin and thealginates. Surfactants generally will serve to suspend the activeingredients, particularly the fat-soluble propionate-enhancingcompounds. Most useful for making suspensions in liquid nonsolvents arealkylphenol polyethylene oxide adducts, naphthalenesulfonates,alkylbenzene-sulfonates, and the polyoxyethylene sorbitan esters. Inaddition many substances, which affect the hydrophilicity, density andsurface tension of the liquid, can assist in making suspensions inindividual cases. For example, silicone anti-foams, glycols, sorbitol,and sugars can be useful suspending agents.

In an aspect, a therapeutic composition comprises non-pathogenic sporesof one or more, two or more, three or more, or four or more Clostridiumspecies selected from the group consisting of Clostridium absonum,Clostridium argentinense, Clostridium baratii, Clostridium botulinum,Clostridium cadaveris, Clostridium carnis, Clostridium celatum,Clostridium chauvoei, Clostridium clostridioforme, Clostridiumcochlearium, Clostridium fallax, Clostridium felsineum, Clostridiumghonii, Clostridium glycolicum, Clostridium haemolyticum, Clostridiumhastiforme, Clostridium histolyticum, Clostridium indolis, Clostridiumirregulare, Clostridium limosum, Clostridium malenominatum, Clostridiumnovyi, Clostridium oroticum, Clostridium paraputrificum, Clostridiumperfringens, Clostridium piliforme, Clostridium putrefaciens,Clostridium putrificum, Clostridium sardiniense, Clostridiumsartagoforme, Clostridium scindens, Clostridium septicum, Clostridiumsordellii, Clostridium sphenoides, Clostridium spiroforme, Clostridiumsporogenes, Clostridium subterminale, Clostridium symbiosum, Clostridiumtertium, Clostridium tetani, Clostridium welchii, and Clostridiumvillosum. In an aspect, a therapeutic composition comprises one or more,two or more, three or more, or four or more non-pathogenic Bacteroidesspecies selected from the group of Bacteroides coprocola, Bacteroidesplebeius, Bacteroides massiliensis, Bacteroides vulgatus, Bacteroideshelcogenes, Bacteroides pyogenes, Bacteroides tectus, Bacteroidesuniformis, Bacteroides stercoris, Bacteroides eggerthii, Bacteroidesfinegoldii, Bacteroides thetaiotaomicron, Bacteroides ovatus,Bacteroides acidifaciens, Bacteroides caccae, Bacteroides nordii,Bacteroides salyersiae, Bacteroides fragilis, Bacteroides intestinalis,Bacteroides coprosuis, Bacteroides distasonis, Bacteroides goldsteinii,Bacteroides merdae, Bacteroides forsythus, Bacteroides splanchnicus,Bacteroides capillosus, Bacteroides cellulosolvens, and Bacteroidesureolyticus. The foregoing Clostridium and Bacteroides can be eithercultured or purified and can be used in combination in a singlecombination for a synergistic effect.

In an aspect, a therapeutic composition comprises purified, isolated, orcultured viable non-pathogenic Clostridium and a plurality of purified,isolated, or cultured viable non-pathogenic microorganisms from one ormore genera selected from the group consisting of Collinsella,Coprococcus, Dorea, Eubacterium, and Ruminococcus. In another aspect, atherapeutic composition comprises a plurality of purified, isolated, orcultured viable non-pathogenic microorganisms from one or more generaselected from the group consisting of Clostridium, Collinsella,Coprococcus, Dorea, Eubacterium, and Ruminococcus.

In an aspect, a therapeutic composition comprises two or more generaselected from the group consisting of Collinsella, Coprococcus, Dorea,Eubacterium, and Ruminococcus. In another aspect, a therapeuticcomposition comprises two or more genera selected from the groupconsisting of Coprococcus, Dorea, Eubacterium, and Ruminococcus. In afurther aspect, a therapeutic composition comprises one or more, two ormore, three or more, four or more, or five or more species selected fromthe group consisting of Coprococcus catus, Coprococcus comes, Dorealongicatena, Eubacterium eligens, Eubacterium hadrum, Eubacteriumhallii, Eubacterium rectale, and Ruminococcus torques.

In one aspect, a pharmaceutical composition is in an anaerobic packageor container. In another aspect, a pharmaceutical composition furthercomprises an oxygen scavenger. In one aspect, a container can be madeoxygen free by e.g., incorporating into the container a built in orclipped-on oxygen-scavenging mechanism, e.g., oxygen scavenging pelletsas described e.g., in U.S. Pat. No. 7,541,091. In another aspect, thecontainer itself is made of an oxygen scavenging material, e.g., oxygenscavenging iron, e.g., as described by O2BLOCK™, or equivalents, whichuses a purified and modified layered clay as a performance-enhancingcarrier of oxygen-scavenging iron; the active iron is dispersed directlyin the polymer. In one aspect, oxygen-scavenging polymers are used tomake the container itself or to coat the container, or as pellets to beadded; e.g., as described in U.S. Pat. App. Pub. 20110045222, describingpolymer blends having one or more unsaturated olefinic homopolymers orcopolymers; one or more polyamide homopolymers or copolymers; one ormore polyethylene terephthalate homopolymers or copolymers; that exhibitoxygen-scavenging activity. In one aspect, oxygen-scavenging polymersare used to make the container itself or to coat the container, or aspellets to be added; e.g., as described in U.S. Pat. App. Pub.20110008554, describing compositions comprising a polyester, acopolyester ether and an oxidation catalyst, wherein the copolyesterether comprises a polyether segment comprisingpoly(tetramethylene-co-alkylene ether). In one aspect, oxygen-scavengingpolymers are used to make the container itself or to coat the container,or as pellets to be added; e.g., as described in U.S. Pat. App. Pub.201000255231, describing a dispersed iron/salt particle in a polymermatrix, and an oxygen scavenging film with oxygen scavengingparticulates.

In preferred aspects, purified fecal microbiota is obtained from acarefully screened, healthy, neurotypical human donor. Microbiota isseparated from fecal material collected from healthy donors, mixed witha cryopreservative, stored as a frozen liquid suspension with thecryopreservative, and thawed prior to administration in liquid form.Based on the route of administration, the purified fecal microbiota canbe provided as fresh, frozen-thawed, or lyophilized live microbiota. Insome cases, purified fecal microbiota is administered to a human subjectin the form of an oral dose. In other cases, purified fecal microbiotais administered in the form of a rectal dose.

In some cases, the dosage form comprises any suitable form of livemicrobiota (fresh, frozen, lyophilized, etc.) and is formulated foradministration to a human subject orally, by nasogastric tube, bycolonoscopy, or anally. In some cases, the dosage is administered as asolution. In other cases, the dosage is administered as solid dosageforms such as, for example, capsules, tablets, powders, and granules. Insuch solid dosage forms, purified fecal microbiota is admixed with atleast one inert excipient (or carrier), a filler or extender (e.g.,starches, lactose, sucrose, mannitol, or silicic acid), a binder (e.g.,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, or acacia), a humectant (e.g., glycerol), a disintegratingagent (e.g., agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, a silicate, sodium carbonate), an absorption accelerators,a wetting agent (e.g., cetyl alcohol or glycerol monostearate), anadsorbent (e.g., kaolin or bentonite), and/or a lubricant (e.g., talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, or mixtures thereof). In the case of capsules andtablets, the dosage forms may also comprise buffering agents.

A tablet comprising purified fecal microbiota can, for example, be madeby compressing or molding the active ingredient, optionally with one ormore additional ingredients. Compressed tablets can be prepared bycompressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets can be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. In exemplary aspects, the dosage form comprises apowder prepared by lyophilization (“freeze drying”), whereby the processinvolves removing water from purified, frozen fecal microbiota atextremely low pressures.

The specific dosage and dosage range that can be used depends on anumber of factors, and the determination of dosage ranges and optimaldosages for a particular patient is well within the ordinary skill ofone in the art in view of this disclosure. It is further understood,however, that the specific dose level for any particular human willdepend upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, gender, anddiet of the human, the time of administration, the route ofadministration, the rate of excretion, any drug combination, and theseverity of any disorder being treated.

In exemplary aspect, purified fecal microbiota is administered to asubject in multiple doses. For example, purified fecal microbiota can beadministered to a subject according to a method provided herein inmultiple doses over a time period of about two days to about eightweeks.

Prior to administration of purified fecal microbiota, any suitableantibiotic can be administered to the subject. In exemplary aspects, theantibiotic is a non-absorbed or minimally-absorbed antibiotic such as,for example, vancomycin or rifaximin. Antibiotics are administered tothe subject via any appropriate delivery route. One of skill in the artcan develop appropriate dose delivery methods. Preferably, theantibiotic is administered to the subject orally. In another aspect, anASD treatment method requires no antibiotic pretreatment. In a furtheraspect, an ASD treatment method requires no bowel preparation or bowelcleansing. In another aspect, an ASD treatment method requires neitherantibiotic pretreatment nor bowel cleansing prior to administering apharmaceutical composition comprising a fecal microbiota preparation.

In some cases, the antibiotic is administered in multiple doses before abowel cleanse is performed. In some cases, administration of theantibiotic is initiated at least seven days (e.g., at least 7, 9, 10,12, 14, 18, or 21 days) before the bowel cleanse. In preferred aspects,the bowel cleanse is preceded by fasting of the human subject.

Following administration of an antibiotic, the subject undergoes a bowelcleanse. In exemplary aspects, the bowel cleanse comprises administeringto the subject a product such as MoviPrep®, a commercial bowel prep forcolonoscopy. Preferably, the bowel cleanse removes residual vancomycinand cleanses the lower gastrointestinal tract.

In exemplary aspects, the method further comprises administering to thesubject a stomach acid suppressant. Stomach acid suppressants, alsoknown as gastric acid suppressants, suitable for use according to amethod provided herein include, without limitation, proton pumpinhibitors (PPIs) and histamine blockers. In some cases, the stomachacid suppressant is Prilosec and is administered to the subject one ormore days in advance of oral administration of purified fecalmicrobiota. In some cases, the stomach acid suppressant is administeredone week prior to oral administration of purified fecal microbiota.

In another aspect, provided herein are unit dosage forms comprisingpurified fecal microbiota. In some cases, unit dosage forms describedherein are provided as part of a kit. Such a kit could include apurified fecal microbiota dosage and, optionally, a delivery device toadminister the composition to the subject or instructions foradministering the dosage to a subject via an appropriate delivery route.In some cases, the dosage form comprises any suitable form of livemicrobiota (fresh, frozen, lyophilized, etc.) and is formulated foradministration to a human subject orally, by nasogastric tube, bycolonoscopy, or anally. As described herein, dosage forms suitable forkits provided herein include, without limitation, liquid solutions,capsules, tablets, powders, granules, and lyophilized forms.

In a further aspect, provided herein is use of a purified compositionfor manufacture of a medicament for treating autism spectrum disorder orfor reducing the severity of one or more symptoms of autism spectrumdisorder.

It will be appreciated that compositions, dosage forms, and medicamentsas described herein include combination pharmaceutical compositions inwhich one or more additional compounds or medications are added to orotherwise co-administered with a purified fecal microbiota composition.

In an aspect, this application provides for the following embodiments:

Embodiment 1

A method for increasing the abundance of one or more gut microorganismsin a subject in need thereof, said method comprising treating saidsubject by administering a therapeutically effective amount of apharmaceutical composition comprising a fecal microbe preparation,wherein said subject exhibits at least a 2-fold increase of theabundance of said one or more fecal microorganisms after said treatmentas compared to before initiating said treatment, wherein said one ormore gut microorganisms are selected from the group consisting ofBifidobacterium, Prevotella and Desulfovibrio.

Embodiment 2

A method for modulating the abundance of one or more gut microorganismsin a subject in need thereof, said method comprising treating saidsubject by administering a therapeutically effective amount of apharmaceutical composition comprising a fecal microbe preparation,wherein said subject exhibits at least a 2-fold change of the abundanceof said one or more fecal microorganisms after said treatment ascompared to before initiating said treatment, wherein said one or moregut microorganisms are selected from the group consisting ofBifidobacterium, Prevotella, Desulfovibrio, Copprococcus, Clostridium,Eggerthella, and Bacteroides.

Embodiment 3

The method of Embodiment 1, wherein said method further comprisesdetermining in said subject a relative abundance of said one or more gutmicroorganisms.

Embodiment 4

The method of Embodiment 3, wherein said relative abundance isdetermined via an assay selected from the group consisting of qPCR,RT-qPCR, clone libraries, DGGE, T-RFLP, ARISA, microarrays, FIFH,dot-blot hybridization, and a DNA hybridization method.

Embodiment 5

The method of Embodiment 3, wherein said relative abundance isdetermined via 16S rDNA-targeted pyrosequencing.

Embodiment 6

The method of claim Embodiment 3, wherein said relative abundance isdetermined via a DNA hybridization assay based on a 16S rDNA sequence.

Embodiment 7

The method of Embodiment 3, wherein said relative abundance isdetermined via detecting one or more proteins or metabolites specific tosaid one or more gut microorganisms.

Embodiment 8

The method of Embodiment 7, wherein said relative abundance isdetermined via an assay selected from the group consisting of2-Dimensional Gel Electrophoresis, 2-Diminsional Difference GelElectrophoresis (2D-DIGE), MALDI TOF-MS, (2D-) LC-ESI-MS/MS, AQUA and1TRAQ.

Embodiment 9

The method of Embodiment 1, wherein said subject has an ASD and saidmethod improves one or more ASD symptoms.

Embodiment 10

The method of Embodiment 9, wherein said one or more ASD symptoms areselected from the group consisting of gastrointestinal (GI) condition,speech, sociability, receptive language, cognition, irritability, mood,anxiety, lethargy, stereotypy, hyperactivity, and play skills.

Embodiment 11

The method of Embodiment 1, wherein said subject exhibits at least a 10%reduction in ASD symptom severity after said treatment as compared tobefore initiating said treatment, and based on an assessment systemselected from the group consisting of Childhood Autism Rating Scale(CARS), Childhood Autism Rating Scale 2—Standard Form (CARS2-ST),Childhood Autism Rating Scale 2—High Functioning (CARS2-HF), AberrantBehavior Checklist (ABC), Social Responsiveness Scale (SRS), andVineland Adaptive Behavior Scale II (VABS-II).

Embodiment 12

The method of Embodiment 1, where said at least 2-fold increase isachieved after 2 or more weeks of initiating said treatment.

Embodiment 13

The method of Embodiment 1, where said at least 2-fold increase ismaintained for at least 8 weeks after discontinuing said treatment.

Embodiment 14

The method of claim Embodiment 1, where said subject exhibits nogastrointestinal (GI) symptom prior to initiating said treatment.

Embodiment 15

The method of claim Embodiment 1, where said subject further exhibitsone or more GI symptoms prior to initiating said treatment.

Embodiment 16

The method of claim Embodiment 15, where said subject exhibits at leasta 50% reduction in GI symptom severity based on the GastrointestinalSymptom Rating Scale (GSRS) after said treatment as compared to beforeinitiating said treatment.

Embodiment 17

The method of claim Embodiment 1, where said method further comprisesadministering an antibiotic to said subject prior to administering saidpharmaceutical composition.

Embodiment 18

The method of claim Embodiment 1, where said method further comprisessubjecting said subject to a bowel cleanse.

Embodiment 19

The method of Embodiment 1, where said at least 10% reduction in ASDsymptom severity is achieved after 2 or more weeks of initiating saidtreatment.

Embodiment 20

The method of Embodiment 11, where said at least 10% reduction in ASDsymptom severity is maintained for at least 8 weeks after discontinuingsaid treatment.

Embodiment 21

The method of Embodiment 11, where said subject exhibits nogastrointestinal (GI) symptom prior to initiating said treatment.

Embodiment 22

The method of Embodiment 11, where said subject further exhibits one ormore GI symptoms prior to initiating said treatment.

Embodiment 23

The method of Embodiment 22, where said subject exhibits at least a 50%reduction in GI symptom severity based on the Gastrointestinal SymptomRating Scale (GSRS) after said treatment as compared to beforeinitiating said treatment.

Embodiment 24

The method of Embodiment 11, where said method further comprisesadministering an antibiotic to said subject prior to administering saidpharmaceutical composition.

Embodiment 25

The method of Embodiment 11, where said method further comprisessubjecting said subject to a bowel cleanse.

Embodiment 26

The method of Embodiment 11, wherein said ASD is selected from the groupconsisting of autistic disorder, pervasive developmental disorder nototherwise specified (PDD-NOS), and Asperger syndrome.

Embodiment 27

The method of Embodiment 1, wherein said pharmaceutical composition isadministered orally.

Embodiment 28

The method of Embodiment 1, wherein said fecal microbe preparation islyophilized.

Embodiment 29

The method of Embodiment 1, wherein said fecal microbe preparationcomprises a non-selective and substantially complete fecal microbiotafrom a single donor.

Embodiment 30

The method of Embodiment 29, wherein said non-selective andsubstantially complete fecal microbiota is supplemented with one or moreviable, non-pathogenic microorganisms selected from the group consistingof Bifidobacterium, Prevotella, Desulfovibrio, Copprococcus,Clostridium, and Bacteroides.

Embodiment 31

The method of Embodiment 1, wherein said fecal microbe preparationcomprises a synthetic fecal composition of predetermined flora.

Embodiment 32

The method of Embodiment 31, wherein said predetermined flora comprisesa preparation of viable flora in proportional content that resembles anormal healthy human fecal flora and comprises no antibiotic resistantpopulations.

Embodiment 33

The method of Embodiment 1, wherein said pharmaceutical composition isadministered as a solid dosage form selected from the group consistingof capsule, tablet, powder, and granule.

Embodiment 34

The method of Embodiment 1, wherein said pharmaceutical composition isformulated as an acid resistant capsule.

Embodiment 35

A method for increasing the phylogenetic diversity of fecal microbiotaof a subject in need thereof, said method comprising administering tosaid subject a therapeutically effective amount of a pharmaceuticalcomposition comprising a fecal microbe preparation, wherein said subjectexhibits at least a two-fold increase of fecal microbiota diversityafter said treatment as compared to before initiating said treatment.

Embodiment 36

The method of Embodiment 35, wherein said subject has an ASD and saidmethod improves one or more ASD symptoms.

Embodiment 37

A method comprising

-   -   a. determining the relative abundance of one or more gut        microorganisms in a subject having an ASD or having an ASD        sibling, and    -   b. administering a therapeutically effective amount of a        pharmaceutical composition comprising a fecal microbe        preparation to achieve at least a 2-fold change of the abundance        of said one or more fecal microorganisms, wherein said one or        more gut microorganisms are from a genus selected from the group        consisting of Clostridium, Bacteroides, Bifidobacterium,        Prevotella, Eggerthella, and Desulfovibrio.

Embodiment 38

The method of Embodiment 37, wherein said subject having an ASD exhibitsno gastrointestinal symptom.

Embodiment 39

The method of Embodiment 37, wherein said subject having an ASD does notexhibit one or more, two or more, three or more, four or moregastrointestinal symptoms selected from the group consisting ofabdominal pain, reflux, indigestion, irritable bowel syndrome, chronicpersistent diarrhoea, diarrhoea, flatulence, constipation, andalternating constipation/diarrhoea.

Embodiment 40

The method of Embodiment 37, wherein said subject having an ASD exhibitsno gastrointestinal symptom selected from the group consisting ofabdominal pain, reflux, indigestion, irritable bowel syndrome, chronicpersistent diarrhoea, diarrhoea, flatulence, constipation, andalternating constipation/diarrhoea.

Embodiment 41

The method of Embodiment 37, wherein said one or more gut microorganismsare from Clostridium, and wherein said Clostridium microorganismsincrease their abundance by at least 2 fold.

Embodiment 42

The method of Embodiment 37, wherein said one or more gut microorganismsare from Bacteroides, and wherein said Bacteroides microorganismsdecrease their abundance by at least 2 fold.

Embodiment 43

The method of Embodiment 37, wherein said one or more gut microorganismsare from Prevotella, and wherein said Prevotella microorganisms increasetheir abundance by at least 2 fold.

Embodiment 44

The method of Embodiment 37, wherein said one or more gut microorganismsare from Desulfovibrio, and wherein said Desulfovibrio microorganismsincrease their abundance by at least 2 fold.

Embodiment 45

The method of Embodiment 1, where said at least 2-fold increase ismaintained for at least 52 weeks after discontinuing said treatment.

Embodiment 46

The method of Embodiment 1, where said at least 2-fold increase ismaintained for at least 98 weeks after discontinuing said treatment.

Embodiment 47

The method of Embodiment 11, where said at least 10% reduction in ASDsymptom severity is maintained for at least 50 weeks after discontinuingsaid treatment.

Embodiment 48

The method of Embodiment 11, where said at least 10% reduction in ASDsymptom severity is maintained for at least 98 weeks after discontinuingsaid treatment.

The disclosure may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of thedisclosure. The following examples are presented in order to more fullyillustrate the preferred aspects of the disclosure and should in no waybe construed, however, as limiting the broad scope of the disclosure.Therefore, the scope of the appended claims should not be limited to thedescription of the aspects contained herein.

EXAMPLES Example 1: Treating Autistic Children Using Microbiota TransferTherapy (MTT)

A clinical study (ClinicalTrials.gov Identifier: NCT02504554) of 20autistic children, ages 7-17, is conducted to evaluate the safety andtolerability of a fecal microbiota-based treatment designed to reducethe symptoms of autism by improving the gastrointestinal microbiotafunction. This treatment includes transfer of purified gut bacteria froma healthy person to children diagnosed as having autism spectrumdisorder. Details of this trial are described in PCT Application No.PCT/US2016/033747 and U.S. application Ser. No. 15/161,654, both filedMay 23, 2016 (both incorporated by reference in their entirety).

The general study design is an open-label clinical trial involving 18children (ages 7-17 years) with ASD who are diagnosed by the AutismDiagnostic Interview-Revised (ADI-R) and have moderate to severegastrointestinal problems. Each child participates in the study for 18weeks in total, a 10 week treatment and a follow-up 8 week observationperiod after the treatment stops. For the fecal material transplant(FMT) treatment, two routes of administration, oral versus rectal, arecompared for the initial dose, followed by a lower maintenance dosagegiven orally for 7-8 weeks.

The protocol is approved by FDA (Investigational new drug number 15886)and the Institutional Review Board of Arizona State University (ASU IRBProtocol #: 00001053). The study is advertised by email to approximately2500 ASD families in Arizona, using the contact list of the AutismSociety of Greater Phoenix and the Autism/Asperger's Research Program atArizona State University. Families with children who meet the studyinclusion and exclusion criteria have a 1-hour individual phone call todiscuss the study. After the phone call, families who sign the parentpermission form and child assent forms are provided with initialquestionnaires to complete. A letter is also sent to them for theirpersonal physician to double-check their medications and for thephysician to be aware of the delivery of the vancomycin, Prilosec, andthe fecal transplant

Beneficial bacteria (a non-selective fecal microbiota preparation) wereprepared from human donor stools. Fecal samples were collected fromcarefully-screened healthy donors (90% of general population rejected)and purified extensively to retain only bacteria. Specifically, themicrobiota is separated from fecal material collected from carefullyscreened, healthy donors, stored in a cryopreservative in a frozenliquid suspension with a cryopreservative, and thawed prior toadministration in liquid form. Each purified sample of beneficialbacteria contained 1000 or more bacterial species. By comparison,standard commercially available probiotics include 1 to 10 bacterialspecies.

Example 2: Subject Recruitment

The study began with a verification of an autism spectrum diagnosisusing the Autism Diagnostic Interview-Revised (ADI-R), which involved aphone interview of the parents with our ADI-R evaluator. The studyphysician assessed general physical health through an initial 30 minutemeeting with participants and an extensive review of the participants'last 2 years of medical records and height/weight/growth charts in orderto check for exclusion criteria. Participant exclusion criteria includeantibiotics in last 6 months and probiotics in last 3 months,single-gene disorder, major brain malformation, tube feeding, severe GIproblems that require immediate treatment (life-threatening), UlcerativeColitis, Crohn's disease, diagnosed Celiac Disease, EosinophilicGasteroenteritis, severely underweight/malnourished, andrecent/scheduled surgeries. None of the neurotypical children arediagnosed with mental disorders including ASD, ADHD, depression, andanxiety, and neurotypical children do not have first-degree relatives ofindividuals with ASD. From participants, initial blood, urine, and stoolsamples are collected and parents are asked to fill in diet diaries oftheir child for one week at the beginning of the study. Participants arerecruited primarily from the greater Phoenix, Ariz. area, but threeparticipants are from outside that area. Neurotypical families arerecruited from friends of the ASD families and professionals who workwith ASD families.

Example 3: Trial Participants

Eighteen autism participants (each from a different family) ages 7-17years with moderate to severe GI problems and moderate to high cognitivefunctioning. Twenty participants are recruited into the study, but twodo not enter the treatment phase of the study before the treatmentstarted. One participant is disqualified due to a change in medication,and one decides not to participate. Characteristics of 18 studyparticipants and their medical history are listed in Table 2. All 18participants that enter the treatment phase complete the 19-week trial.The post-treatment data presented herein is collected for 13 of these 18participants. In addition, 20 age- and gender-matched neurotypicalchildren from 13 families (6 families had 1 neurotypical participant,and 7 families had 2 neurotypical participants) are also recruited.These 20 neurotypical children are monitored for 18 weeks but nottreated.

TABLE 2 Characteristics of study participants and their medical history.Children Neurotypical with ASD children p-value Total Number 18 20Male/Female 16/2 18/2 Age 11.0 +/− 2.7  11.1 +/− 2.5 n.s. BMI 19.9 +/−5.4  18.1 +/− 3.4 n.s. GSRS 4-point scale (sum of all 15 28.1 +/− 4.3 18.8 +/− 4.0 P < 0.001 items, minimum score for no symptoms is 15) Bornby C-Section 61% 16%  P < 0.01 Number of months of breast- 3.3 +/− 3.9 9.3 +/− 7.8 P < 0.01 feeding exclusively (no formula) % usingnon-standard formula 39% 8% P < 0.05 (soy or other) Food allergy(moderate or severe) 56% 5% P < 0.01 Other allergies 44% 10%  P < 0.01(moderate or severe) Eczema 56% 5% P < 0.01 Fiber consumption - child8.9 +/− 4.3 11.8 +/− 4.9 P = 0.07 Fiber consumption - mother 6.7 +/− 3.910.5 +/− 4.5 P = 0.02 Oral antibiotic use during first 4 4.6 +/− 5.2 4.1 +/− 6.0 n.s. years of life (number of rounds)

Example 4: Trial Protocol

The participants are given oral vancomycin (a non-absorbable broadspectrum antibiotic that stays in the GI tract) for 2 weeks to reducelevels of pathogenic bacteria, and then 1 day of low-volume colonoscopyprep MoviPrep® (a drink that flushes the bowels, to remove mostremaining gut bacteria and vancomycin) to clear the residual vancomycinand feces. The vancomycin is intended to kill off harmful bacteria, thefasting is intended to remove any remaining bacteria and to minimizeother luminal fecal material, and the colon cleanse helps to remove thevancomycin and cleanse the lower GI Tract.

Following vancomycin treatment and bowel cleanse, participants receivedeither 2 days of high dose oral Microbiota Transfer Therapy (MTT, mixedin a chocolate milk, milk substitute, or juice) (dosage of 2.5×10¹² CFUper day) or a single dose of rectal MTT (dosage of 2.5×10¹² CFU for onegiven similar to an enema). The rectal dose is administered under thedirect supervision of the study physician, and the first oral dose issimilarly administered in the presence of the physician. Participantsare randomly assigned to either the oral or rectal route ofadministration. If one administration route is not tolerated, or if thefamily preferred the other route, then participants had the option oftrying the other route. For the participants with initial oral dose, alower oral maintenance dose (2.5×10⁹ CFU) is followed for 8 weeks rightafter the major oral initial dose. Whereas, the major rectal initialdose is followed by waiting period of 1 week followed by a lower oralmaintenance dose (2.5×10⁹ CFU) for 7 weeks. The maintenance SHGM dose isself-administered orally every day up to week 10. After treatment isstopped, participants were monitored for another 8 weeks. Participantsare then monitored for a 2-year follow-up.

Prilosec (omeprazole) is administered daily to reduce stomach acid andthereby increase viability of the MTT, starting on the 12th day of oralvancomycin treatment and continuing until the end of the maintenancedose. Table 3 provides a general treatment timeline.

TABLE 3 MTT Treatment Timeline Summary. Time (Day) Initial oraladministration Initial rectal administration Day 1-14 Vancomycin* Day12-74 Prilosec* Day 15 MoviPrep* Day 16 Major oral dose of MTT** Majorrectal dose of MTT** Day 17 Major oral dose of MTT — Day 18-24 Lowermaintenance oral — dose of MTT Day 25-74 Lower maintenance oral dose ofMTT*** Day 75-130 No treatment, observation period Day 714-834 Notreatment, observation period *Vancomycin: 40 mg/kg P.O. per day,divided into three doses, not to exceed 2 gm per day; Prilosec: 20 mg POQD; MoviPrep: Standard kit is used with half the dosage beingadministered at approximately 10 am and the other half at 4 pm on dayfifteen only, to cleanse the bowel of vancomycin and feces. The dosagevaries proportionately based on the body mass. **Initial oral route: Thedosage for the first 2 days will be 8.3 × 10¹¹ cells, t.i.d, for a totaldaily dose of 2.5 × 10¹² cells/day, for Day 16 and 17 only; Initialrectal route: 2.5 × 10¹² cells, 1x (Day 16 only) ***Maintenance dose:2.5 × 10⁹ cells, 1x/day P.O.

Example 5: Fecal Microbiota Preparation Used for MTT

A human microbiota preparation, which comprises a highly purifiedstandardized extract from human feces (also called Standardized HumanGut Microbiota (SHGM)) is used. This is a full-spectrum product,containing all the bacteria present in the gut of very healthy donors.First, donors are carefully screened using an extensive healthquestionnaire and extensive medical testing to ensure optimal GI andoverall health; the screening process is so rigorous that 90% of donorsare eliminated, leaving only the 10% healthiest portion of thepopulation. The donated material is then extensively filtered andstandardized, following FDA Good Manufacturing Processes (GMP). Thefinal product is liquid form which can be frozen, and is proven to behighly effective for treating C. difficile (Hamilton et al., Am JGastroenterol. 2012 May; 107(5):761-7). The SHGM is stored in −80° C.freezers and then delivered to families on dry ice every week during thestudy. Families are instructed to keep the SHGM in a container with dryice, and thaw it shortly before use.

Two different doses of SHGM are used; the high major dose and a lowermaintenance dose. The high-dose SHGM is at a daily dosage of 2.5×10¹²cells. The rationale for two days of high dose is that after theMoviPrep and 1-day fast is presumably the most critical time in which toprovide new beneficial bacteria. The low-dose SHGM is at a dosage of2.5×10⁹ cells.

Example 6: Toleration of Study Medications

Vancomycin: The vancomycin is associated with two types of minor adverseevents. One child develops an allergic rash upon administration of oralvancomycin, but they are switched to vancomycin without orange flavoringand the rash disappeared. Twelve of the 18 children had a behavioralreaction to the vancomycin, starting 1-4 days after the start of thevancomycin, and lasting 1-3 days in most cases, although 1 participanthad symptoms lasting for 3 weeks. In 7 cases, the symptoms are mild tomoderate increase in hyperactivity, and in 5 cases the symptoms are mildto moderate increase in tantrumming/aggression. After these behavioralsymptoms disappeared, GI symptoms and autism symptoms began improving.Similar results are reported in a previous study (Sandler, 2000), andparents of the study subjected had been informed to expect this. Thereaction may be due to release of bacterial toxins as the vancomycinkills off harmful bacteria.

Prilosec: This is generally well-tolerated.

MoviPrep®: Many children had difficulty consuming this medication due totaste.

Rectal administration of Microbiota Transfer Therapy (MTT): This issurprisingly well-tolerated by 6 of 6 recipients.

Oral administration of high-dose MTT: This is well-tolerated by 12 of 13recipients, but 1 participant experienced vomiting and is switched tothe rectal route.

Oral administration of maintenance dose MTT: This is well-tolerated byall participants.

CBC/ChemPanel: There are no major concerns regarding changes in CompleteBlood Count (CBC) or blood chemistry panel (CBC). The following minorchanges are observed. There is a 5% decrease in potassium (p=0.01) frombeginning to end of treatment, but all levels remain in the normalrange. After the vancomycin (2nd week of study), there is a 8% increasein platelets (p=0.03). Four subjects had elevated levels at start, andonly 2 had elevated levels after vancomycin. There is a 26% drop inblood urea nitrogen (BUN) (p=0.002), but all stayed in normal range.There is a 6% increase in albumin to globulin (A/G) ratio (p=0.03), with1 slightly elevated. There is a 17% increase in aspartate aminotransferase (AST) (p=0.01), but all remained in normal range. There is a24% increase in alanine amino transferase (ALT) (p=0.003), where 1remained elevated and 2 became slightly elevated. All of these values(platelets, BUN, A/G, AST, ALT) returned to similar to baseline at the3rd and 4th tests. Slight changes (1-2%) in red blood cell indices (Meancorpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Meancorpuscular hemoglobin concentration (MCHC), and Red cell distributionwidth (RDW)) are observed.

Example 7: Adverse Effects

Children with ASD experienced temporary adverse effects at the beginningof vancomycin treatment. As listed in Table 4, one participant among the18 children with ASD (5%) developed an extensive rash, but the rashdisappeared when vancomycin is switched from a natural orange flavor toan unflavored form. Within 1-4 days after the start of the vancomycin,12 children with ASD had a temporary behavioral reaction to thevancomycin either involving hyperactivity (7 out of 12 cases; 39%) orTantrums/Aggression (5 out of 12 cases; 28%). The symptoms lasted 1-3days in most cases, except for one participant that had symptoms lastingfor 3 weeks. After the symptoms disappeared, GI symptoms and behavioralsymptoms began improving, which is similar to what Sandler et al.,Journal of Child Neurology 15, 429-35, (2000) reported in their oralvancomycin therapy for children with autism. Only one participant doesnot tolerate the initial high-dose oral SHGM (nausea/vomiting) and isswitched to initial rectal administration.

TABLE 4 Adverse effects. Adverse effect % adverse effects Rash 5% (dueto natural orange flavor in vancomycin) Hyperactivity 39%* (temporary:start of vancomycin only) Tantrums/Aggression 28%* (temporary: start ofvancomycin only) Nausea/vomiting 5% (due to high-dose SHGM) *Theseverity of symptoms ranged from mild to moderate.

Example 8: Assessments of Gastrointestinal Symptoms

Gastrointestinal Symptom Rating Scale (GSRS) is an assessment of GIsymptoms during the previous week, based on 15 questions, which are thenscored in 5 domains: Abdominal Pain, Reflux, Indigestion, Diarrhea, andConstipation. A score is reported for each domain based on the averagewithin the questions in that domain. The original GSRS used a 4-pointscale, but a revised version is used which included 7-point Likert scalewhich also has simpler language. The GSRS is assessed on days 0, 7, 14,21, 28, 35, 42, 56, 74, and 130. One of ordinary skill in the artunderstands that GSRS is only one way to assess GI symptoms. Othersimilar tools can be used or designed to evaluate GI symptoms.

Daily Stool Records (DSR) are collected at baseline for two weeks, dailyduring the treatment phase, and the last two weeks of the observationperiod. These records included a rating of the stool using the BristolStool Form scale (1=very hard, 7=liquid).

Example 9: Assessments of Autism and Related Symptoms

Autism Diagnostic Interview-Revised (ADI-R) is a 2-hour structuredinterview and is one of the primary tools used for clinical diagnosis ofautism and autism spectrum disorders. It is not designed to be a measureof autism severity, but higher scores are generally consistent with moresevere symptoms. The ADI-R is used to verify the diagnosis of ASD foradmission into the study.

Parent Global Impressions—III is introduced here as an expanded versionof the PGI-R. See Adams et al., Effect of a Vitamin/Mineral Supplementon Children with Autism, BMC Pediatrics, 11:111(2011). The PGI-IIIevaluates changes in 17 areas (see FIG. 13), and overall, using a7-point scale ranging from “much worse” to “much better”. An “AverageChange” is computed by computing the average in all 18 scores of thePGI-2-Final. This tool is chosen because it was found that it is morereliable to ask parents directly about observed changes than to havethem estimate symptom severity at beginning and end and then compute adifference. Also, the use of a 7-point scale to detect changes seems toyield a high sensitivity to changes.

Childhood Autism Rating Scale (CARS) is a 15-item scale that can be usedto both diagnose autism and ASD and to assess the overall severity ofsymptoms. The CARS assessment is done subsequent to the ADIR assessmentby the same evaluator.

Aberrant Behavior Checklist (ABC) assesses problem behaviors in fiveareas common in children with ASD, including irritability, lethargy,stereotypy, hyperactivity, and inappropriate speech.

Social Responsiveness Scale (SRS) is a 65-item scale that assessessocial impairments, a core issue in autism, including social awareness,social information processing, capacity for reciprocal socialcommunication, social anxiety/avoidance, and autistic preoccupations andtraits. See Constantino et al., Validation of a brief quantitativemeasure of autistic traits: comparison of the social responsivenessscale with the autism diagnostic interview-revised. J Autism Dev Disord.2003 August; 33(4):427-33.

Vineland Adaptive Behavior Scale II (VABS-II) is a measure of thefunctioning level in four different domains: Communication, Daily LivingSkills, Socialization, and Motor Skills, and 11 sub-domains. The rawscores are converted into an age equivalent score. It complements theABC, which assesses problem behaviors. See Sara et al., VinelandAdaptive Behavior Scales, Second Edition (Vineland™-II), PearsonPublishing, 2005.

The GSRS and PGI-R3 are assessed on days 0, 7, 14, 21, 28, 35, 42, 56,74, 130, and an average of analysis taken on days 714-834. The StoolRecord is assessed every day during the treatment. The CARS, ABC, andSRS are assessed at baseline, at the end of treatment, and at the end ofthe observation period. The VABS-II is assessed at baseline and at theend of the observation period, and at the 2-year follow-up only, becauseit is lengthy and believed to be less sensitive to short time periodssince it assesses changes in specific adaptive skills. The CARS isassessed by a professional evaluator, and the GSRS, PGI-R2, ABC, SRS,and VABS-II are assessed by parents.

Example 10: Initial Observations

GI Symptoms:

During the 2 weeks of vancomycin and then 8 weeks of beneficialbacteria, there is a rapid improvement in GI symptoms in most children.At the end of treatment there is an 82% reduction in average scores onthe Gastrointestinal Symptom Rating Scale (GSRS) (FIG. 1 and FIG. 3). Asshown in FIG. 2 and FIG. 5, roughly equal decrease in all 4 GSRSsubscale areas (abdominal pain, indigestion, diarrhoea, constipation).There is no change in the reflux subscale because none of the childrenhad a significant reflux problem. Sixteen of 18 children had a 70% orgreater reduction, 1 had a 30% reduction, and 1 exhibited no change.Similar results are obtained for both the rectal-administration groupand the oral-administration group.

Autism Symptoms:

By the end of the treatment phase, the parents rated their children'sautism symptoms on the Overall scale of the Parent Global Impressionsas: Much Better—4; Better—8; Slightly Better—5; Little/No change—1. Thelargest improvements are in GI, speech, sociability, receptive language,cognition, irritability/mood, anxiety, and play skills (FIG. 3). For theChildhood Autism Rating Scale (CARS) rated by our experienced evaluator,there is a 22% decrease in the CARS scores, p<0.001, which is consistentwith the observations by the parents. For the Aberrant BehaviorChecklist (ABC), there is a 27% reduction in the total score, p=0.001(FIG. 4). Similar results are obtained for both therectal-administration group and the oral-administration group.

Post-Treatment:

Among the first 5 participants that completed the 8-week post-treatmentobservation period, after two months of receiving no treatment, onaverage no change in improvements of GI symptoms is observed (73%reduction in GSRS at end of treatment vs. start; 71% reduction after 8weeks of no treatment vs. start). With respect to post-treatment autismsymptoms, PGI-Scores continued to improve over those collected at theend of treatment, with medium to large improvements in 3 participantsand no detected change in 2 participants. (FIG. 7). With regard topost-treatment CARS scores, these 5 children had a 16% decrease in CARSscores at the end of treatment, and a 25% decrease compared to baselineat the end of the no-treatment (observation) period. So, there appearedto be a surprising continued improvement in symptoms even aftertreatment stopped.

These data demonstrates a 22% reduction in autism severity scoresassigned using the Childhood Autism Rating Scale (CARS) after only 10weeks of the combined therapy (FIG. 3). The degree of improvement on theCARS does not appear to correlate with age (FIG. 6). This suggests thatthe treatment is useful for both younger children and adults.Furthermore, the degree of improvement on the CARS does not correlatewith initial GSRS score (FIG. 8). This suggests that the treatment ishelpful to those with mild GI symptoms as well as those without GIsymptoms. In other words, the treatment appears to be effective toreduce autism symptoms regardless of the presence or absence of GIsymptoms. This observation is consistent with data reported in ourprevious study (Kang et al., PLOS One 8(7):e68322 (2013)), from which itis concluded that children with ASD had a low diversity of gut bacteriathat is independent of their gastrointestinal symptoms.

Example 11: Trial Outcome and Analysis

Clinically, this study is broadly successful. First, all ASDparticipants completed the 18-week study. Second, GI symptoms, asassessed by the Gastrointestinal Symptom Rating Scale (GSRS),significantly improved for abdominal pain, indigestion, diarrhea, andconstipation, such that the average GSRS score dropped 82% from thebeginning to end of treatment and remained improved (77% decrease frombaseline) at 8 weeks after treatment stopped (two-tailed paired t-testt=−9.45, p<0.001, t=−7.64, p<0.001, respectively) (FIG. 9, panel a). Asteady and large degree of improvement in most areas of GSRS evaluationincluding abdominal pain, indigestion, diarrhea, and constipation (FIG.10, panel a) are observed. There is little change in reflux since nochildren had significant reflux at the start of the study. Notably, twoseemingly opposite GI symptoms-diarrhea and constipation-responded tothe MTT treatment effectively.

Similarly, the Daily Stool Record (DSR), showed significant decreases inthe number of days with abnormal or no stools, and those improvementsremained after 8 weeks of no treatment (Table 5, FIG. 10, panel b). TheDaily Stool Record (DSR) is collected and averaged over two weeks inorder to assess changes in stool hardness/softness during the study.Overall, a significant decrease is observed in “% days of abnormalstool” that combines % days of hard, soft/liquid, and no stool, from 62%to 34% (p=0.001) during the 10-week MTT treatment (Table 5 and FIG. 10,panel b). The improvements remained stable for the following 8 weeksduring the observation period. In detail, both “% days of hard stools”(type 1 or 2) and “% days of soft/liquid stools” (type 6 or 7)significantly decreases during the 10-week MTT treatment, but thedecrease in “% days of no stool” was not significant. (Table 5).

TABLE 5 Percent days of no stool, stool hardness and softness based onthe daily stool record (DSR) and the Bristol Stool Form Scale. 8 weeksTreatment after Baseline end p-value treatment p-value No stool 33% 26%0.27 26% 0.38 Hard stool 19% 6% 0.04 3% 0.01 (type 1 or 2) Soft/liquidstool 10% 2% 0.05 3% 0.11 (type 6 or 7) Abnormal stool 62% 34% 0.000732% 0.001 (in total of hard, soft/liquid/, no stool)

Third, there are only temporary adverse effects (primarily mild tomoderate hyperactivity and tantrums/aggression) from vancomycintreatment (Table 4), but no major changes in blood chemistry orlong-term adverse effects.

Beyond these GI improvements, ASD-related behavior also improvedfollowing MTT. First, the Parent Global Impressions (PGI-R) assessment,which evaluates 17 ASD-related symptoms, revealed significantimprovement during treatment and no reversion 8 weeks after treatmentended (FIG. 9, panel b). Further, a significant negative correlationbetween GSRS and PGI-R (Spearman correlation test showed r=−0.59 andp<0.001, FIG. 11) suggests that GI symptoms impact ASD behaviors, andthat these can be altered via MTT. By the end of the MTT treatment atweek 10, the parents rated the change in their children's autismsymptoms using the PGI-R, and the largest improvements are in the GIsubscore among 17 subscales and “Overall autism/related symptoms” of thePGI-R (FIG. 12). Specifically, the overall scale of PGI-R is rated asMuch Better: n=4 (22%); Better: n=8 (44%); Slightly Better: n=5 (28%);Little/No change: n=1 (6%). The improvement in the other subscales isshown in FIG. 12.

Second, the Childhood Autism Rating Scale (CARS), which rates core ASDsymptoms, decreases by 22% from beginning to end of treatment and 24%(relative to baseline) after 8 weeks of no treatment (p<0.001, FIG. 9,panel c).

Third, ASD-afflicted children saw improvement in their scores in theSocial Responsiveness Scale (SRS), which assesses social skill deficits(FIG. 9, panel d), and the Aberrant Behavior Checklist (ABC), whichevaluates irritability, hyperactivity, lethargy, stereotypy, andaberrant speech (FIG. 9, panel e). FIG. 10, panel c also shows a moredetailed breakdown of ABC analysis to assess treatment effects onbehaviors common in children with ASD: irritability, lethargy,stereotypy, hyperactivity, and inappropriate speech. In all fivesub-scales, a significant reduction at the end of treatment is observed.

Fourth, the Vineland Adaptive Behavior Scale II (VABS-II) scoring foundthat the average developmental age increased by 1.4 years (p<0.001,VABS-II) and across all sub-domain areas (FIG. 13) during MTT; thoughthe final VABS-II score is still lower than their chronological age.VABS-II is a measure of the functioning level in four different domains:Communication, Daily Living Skills, Socialization, and Motor Skills,based on 11 sub-domains. Among 11 subscales, Fine and Gross Motor skillsare excluded, since these two subscales for the Vineland are onlycalculated up to 6.8 years and most children with ASD improved near tothe limit of the scale. The other 9 subscales and their average arecompared between the baseline and at the end of the study. The MTTtreatment resulted in a significant increase in average developmentalage, from 5.4 years at baseline to 6.8 years at the end of the study(p<0.001). A gain of 1.4 years within 18 weeks of the study is asubstantial increase, but they still remained below their chronologicalage of 10.9 years. Significant improvements are also observed in all 9subscale areas with the largest gains in Interpersonal Skills (2.2years), Personal Living Skills (1.8 years), and Coping Skills (1.7years) (FIG. 13). It is notable that the major impairments in ASD,namely Receptive language, Expressive language, and Interpersonalskills, are among the lowest initial scores, with initial developmentalages of 3.1 years, 4.5 years, and 2.9 years, respectively; all threeareas had substantial improvements of 1.3, 1.1, and 2.2 years,respectively.

Finally, the MTT appears to be beneficial across both younger and olderindividuals (no significant correlations between age and GSRS or CARSimprovement) and whether the initial MTT dose is received orally orrectally. Under our sample size, no difference is observed in efficacyof treatment or clinical outcomes whether MTT is initially administeredrectally or orally.

Together these findings show that MTT is well-tolerated across anage-diverse cohort of 18 ASD-afflicted children. MTT is also effectiveas it led to significant improvements in both GI- and behavior-relatedsymptoms that are sustained at least 8 weeks after treatment.

Example 12: Gut Microbiome Analysis

Changes in the bacterial diversity of gut samples from ASD patients orneurotypical participants are evaluated for correlates to the observedclinical improvements. Briefly, bacteria are surveyed by standardsequencing of 16S rRNA gene PCR-amplicons and definition of operationaltaxonomic units (OTUs). Abundance profiles of microbial OTUs are thenstatistically evaluated for changes over time.

It is observed that gut microbiota are significantly less diverse in ASDchildren than controls at baseline (FIG. 14, panel a; one-tailed t-testt=−2.25, p=0.015, n=18). After MTT, however, bacterial diversityincreased in ASD children (FIG. 14, panel a; paired two-tailed t-testt=4.56, p=0.0003, n=18) such that median richness at week 18 isstatistically indistinguishable across the ASD and control groups (FIG.14, panel a; two-tailed t-test t=0.47, p=0.64, n=18). This increase isobserved in nearly all individuals including one of the twonon-responders (subjects whose GI symptoms do not improve) (FIG. 14,panel b). Higher gut microbiota richness is associated with healthystates. This is presumably, without being bound to any scientifictheory, due to resilience afforded by higher functional redundancy.

It is also observed that the donor microbiota at least partiallyengrafted in the recipient gut. Specifically, the unweighted UniFracdistance, a qualitative measure of microbiota phylogenetic similarity,between the recipient gut and their most recent donor samplesignificantly decreases over time (FIG. 14, panel c; p<0.01 at threeweeks and p<0.001 at 10 and 18 weeks), and remained more similar to thedonor's microbiota 8 weeks after treatment stopped. By the end oftreatment (week 10) and even 8 weeks after treatment stopped (week 18),the distance between the recipient and the donor microbiota is less thannormal interpersonal microbiota variation (FIG. 14, panel c). Sinceexogenous bacteria are usually washed out within a month in the humangut, these signatures of engraftment indicate that MTT overcame“colonization resistance”. The degree of engraftment varied acrossparticipants, which potentially reflects variation in starting gutmicrobiota and diets.

It was further observed that, consistent with a lack of difference intreatment efficacy between rectal and oral administration, receiving MTTeither orally or rectally does not lead to different patterns inpatients' gut microbiota diversity changes. (FIG. 15).

Procedures for microbiome 16S rDNA library preparation, sequencing, andanalysis are summarized below.

Parents are asked to collect stool samples from their child onapproximately days 0, 21, 70, and 126, and to collect fecal swabsbi-weekly on days 0, 14, 21, 28, 42, 56, 70, 84, 98, 112, and 126. Thestool samples are analyzed to determine the types and amounts of gutmicrobiota present. For safety tests, blood and urine samples are alsocollected on approximately days 0, 19, 33, and 74. During the study, theparticipants met with the physician for an initial physical evaluation(including review of medical history) and following evaluations on days16, 30, and 74. The physician had a phone consult with families on days7, 21, 42, and 130, and more frequently if adverse symptoms occurred, orif families had any questions. Neurotypical participants do not receiveany treatment. They simply provided stool samples (at weeks 0 and 19),and swab samples every 2 weeks.

Microbial DNA from stools, swabs, and donor samples is extracted withPowerSoil® DNA Isolation Kit (Mobio Carlsbard, Calif.). 16S rRNA libraryfor MiSeq Illumina platform, is constructed according to the protocolfrom Earth Microbiome Project. The barcoded primer set 515f-806r forpair-ended sequencing of the 16s rRNA V4 region is used (Caporaso et al.ISME Journal, 6:1621-24 (2012)). Library preparation and sequencing workis performed at the Microbiome Analysis Laboratory in the Swette Centerfor Environmental Biotechnology. The used primers amplify both bacterialand archaeal 16S rRNA. No changes specific to archaea are observed.

Sequencing data are analyzed using QIIME 1.9.1 (Caporaso et al. NatureMethods 7:335-36 (2010)), biom-format version 2.1.5 (McDonald et al.Gigascience, 1:6 (2012)), vsearch version 1.7.0(https://github.com/torognes/vsearch), SSU-ALIGN 0.1 (Nawrocki,Bioinformatics, 25:1335-37 (2009)) and FastTree (Price et al. MolecularBiology and Evolution, 26:1641-50 (2009)). Sequence quality control anddemultiplexing is performed using QIIME's split libraries fastq.py withdefault parameters as described in (Bokulich et al, Nature Methods,10:57-U11 (2013)) on a per run basis. The sequences are combined acrossruns by merging the resulting files using the cat Unix command, andsequences are clustered into OTUs at sequence similarities of 100% and97%. All commands that are applied for these analyses are provided inthe GitHub repository available athttp://github.com/gregcaporaso/autism-fmtl.

Microbiome profiles across even sampling depths and OTU percent identitythresholds are analyzed to achieve the maximum resolution in OTUs. Thiscan reveal important differences in microbiomes that are apparent onlyby observing differences in presence, absence, and abundance of closelyrelated taxa that may be grouped into single OTUs at the commonly used97% similarity threshold. Both 100% and 97% OTUs are therefore defined.The analysis here focused on the 100% OTUs, but also compared featuresof the microbiome to those computed based on 97% OTUs for validation.

To validate the method, it is further confirmed that the measures ofcommunity richness and composition used in our study are correlatedbetween the 100% and 97% OTUs, such that if analyses are insteadperformed on 97% OTUs, similar results would be achieved. Specifically,Faith PD (Pearson r=0.97038, p<0.001, n=569), unweighted UniFrac (Mantelr: 0.92999, p<0.001, n=569), and weighted UniFrac (Mantel r: 0.76651,p<0.001, n=569) are all highly correlated across the two OTU clusteringthresholds, suggesting that the same conclusions would be drawn witheither approach. Small differences are expected, as there are many more100% OTUs than 97% OTUs.

Similarly, the richness and composition metrics are computed at evensampling (rarefaction) depths of 5721 and 10,040 on our 100% OTU data.The lower depth allowed for maximizing the number of samples that couldbe analyzed, and comparison to the higher depth allows for confirmingthat results would be similar if more sequences are retained. Faith PD(Pearson r=0.99738, p<0.001, n=548), unweighted UniFrac (Mantel r:0.97296, p<0.001), and weighted UniFrac (Mantel r: 0.99953, p<0.001) areall highly correlated across sampling depths, suggesting that the sameconclusions would be drawn based from either sampling depth.

A customized pipeline is used to compute 100% OTUs. First, sequences areclustered into 100% OTUs with vsearch. The resulting data are loadedinto a BIOM table using the biom from-uc command. OTUs that occurred inonly one sample are filtered from the table for computationalefficiency. OTU representative sequences are aligned with ssu-align, andhigh entropy positions are filtered with ssu-mask. A phylogenetic treeis built using FastTreeMP for phylogenetic diversity analyses. Taxonomyis assigned to the representative sequences using QIIME's RDP Classifierwrapper against the Greengenes 13_5 reference database. Alpha and betadiversity analyses are performed using QIIME'score_diversity_analyses.py, at rarefaction depths of 5721 (to retain asmany samples as possible), and 10,000 to confirm that results aresimilar with more sequences per sample. Meanwhile, 97% OTUs are computedusing QIIME's pick_open_reference_otus.py with the Greengenes 13_5reference database and default parameters. Alpha and beta diversityanalyses are performed using QIIME's core diversity analyses.py at ararefaction depth of 5721.

Example 13: MTT Changes the Abundance of Selected Bacterial Genera inASD Patients

Several bacterial genera changed their abundance significantly followingMTT. These genera include Bifidobacterium (FIG. 14, panel e), Prevotella(FIG. 15, panel f) and Desulfovibrio (FIG. 15, panel g) which bothincreased in abundance, and Bacteroides (FIG. 15, panel h), whichdecreased in abundance. Additional information for taxonomic changesaccompanying MTT is provided in Table 6.

Previous reports asserted that Bifidobacterium are under-represented inASD-afflicted children (Buie et al., Pediatrics 125, S1-S18 (2010);Bravo et al., PNAS 108:16050-55 (2011)). This is also observed in thisstudy at baseline (two-tailed Mann-Whitney U-test p<0.05), but followingMTT, Bifidobacterium significantly increased 4-fold to a median finalrelative abundance of 1.0% (FIG. 14, panel e), this suggests strongengraftment by these microbes in particular.

Prevotella are observed to be under-represented in ASD-afflictedchildren (at baseline median neurotypical and ASD abundances are 0.022%and 0.005%, respectively), but following MTT Prevotella increased250-fold to a median final abundance of approximately 1.3% (Wilcoxonstatistic: 40, p=0.024, FDR-corrected p=0.075). Along with Prevotella,Desulfovibrio also increased ˜150-fold after MTT from baseline to 8weeks after treatment. Without being bound to any scientific theory,this suggests strong engraftment by this microbe in particular. BothDesulfovibrio and Prevotella are on average more abundant in MTTrecipients following treatment than in the donor samples, illustratingthat the transferred microbiota adapts in its new host, but also thatMTT changes the gut environment in a way that is more hospitable torecruit new commensal bacteria. Taken together, these data suggest thatMTT successfully shifts the ASD microbiota toward that of age/gendermatched healthy controls and to that of their donors.

TABLE 6 Selected fecal bacterial genera change their abundance inresponse to MTT. This table shows the abundance of different genera ofbacteria at baseline and 8 weeks after MTT for the ASD group. Thep-values show which results are statistically significant withoutcorrection for multiple hypothesis testing, and the q values show whichresults are significant (q < 0.05) or marginally significant (q < 0.10)after correction for multiple hypothesis testing. The following generashow a significant increase: Bifidobacterium, Prevotella, andDesulfovibirio; The following genus showed a marginally significantdecrease: Bacteroides and Eggerthella. Fold Fold Donor NeurotypicalInitial ASD donor final median median median Taxonomy enrichmentenrichment abundance abundance abundance k_Bacteria; p_Proteobacteria;c_Betaproteobacteria; o_Burkholderiales; inf inf 2.10E−05 0 0f_Oxalobacteraceae k_Bacteria; p_Proteobacteria; c_Betaproteobacteria;o_Burkholderiales; inf inf 0 0 0 f_Oxalobacteraceae; g_Oxalobacterk_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; inf inf0.003035093 0 0 f_S24-7; g_(—) k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; inf inf 0.003035093 0 0 f_S24-7k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 2.0018213756.148439423 0.005964801 0.007595364 0.002979687 f_Ruminococcaceae;g_Gemmiger k_Bacteria; p_Proteobacteria; c_Betaproteobacteria;o_Burkholderiales; inf inf 0.000505136 0 0 f_Comamonadaceae k_Bacteria;p_Proteobacteria; c_Deltaproteobacteria; 26.54064356 147.6037390.001180314 3.36E−05 4.45E−05 o_Desulfovibrionales;f_Desulfovibrionaceae; g_Desulfovibrio k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; inf inf 0 0 0 f_Ruminococcaceae;g_Anaerofilum k_Bacteria; p_Proteobacteria; c_Betaproteobacteria;o_Burkholderiales; inf inf 0.00065189 0 0 Other; Other k_Bacteria;p_Proteobacteria; c_Betaproteobacteria; o_Burkholderiales; inf inf0.00065189 0 0 Other k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; inf inf 0 4.21E−05 0 f_Ruminococcaceae;g_Subdoligranulum k_Bacteria; p_Proteobacteria; c_Alphaproteobacteria;inf inf 0.00035831 0 0 o_RF32 k_Bacteria; p_Proteobacteria;c_Alphaproteobacteria; inf inf 0.00035831 0 0 o_RF32; f_; g_(—)k_Bacteria; p_Proteobacteria; c_Alphaproteobacteria; inf inf 0.000358310 0 o_RF32; f_(—) k_Bacteria; p_Lentisphaerae; c_[Lentisphaeria];o_Victivallales inf inf 0 0 0 k_Bacteria; p_Lentisphaerae;c_[Lentisphaeria] inf inf 0 0 0 k_Bacteria; p_Lentisphaerae inf inf 0 00 k_Bacteria; p_Lentisphaerae; c_[Lentisphaeria]; o_Victivallales; infinf 0 0 0 f_Victivallaceae k_Bacteria; p_Actinobacteria;c_Coriobacteriia; o_Coriobacteriales; 0 4.081749272 0 5.23E−05 4.89E−05f_Coriobacteriaceae; g_Adlercreutzia k_Bacteria; p_Proteobacteria;c_Deltaproteobacteria; 1.313603645 3.843000937 0.002789742 0.0014618410.002123732 o_Desulfovibrionales; f_Desulfovibrionaceae k_Bacteria;p_Proteobacteria; c_Deltaproteobacteria; 1.313603645 3.8430009370.002789742 0.001461841 0.002123732 o_Desulfovibrionales k_Bacteria;p_Proteobacteria; c_Deltaproteobacteria 1.313603645 3.8430009370.002789742 0.001461841 0.002123732 k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; inf inf 0.000105144 0 0f_[Odoribacteraceae]; g_Butyricimonas k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0 0 0 9.72E−05 0.000225701f_Lachnospiraceae; g_Ruminococcus k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 6.827225474 2.419218284 0.0070009770.002181422 0.00102545 f_Peptostreptococcaceae; g_(—) k_Bacteria;p_Proteobacteria; c_Alphaproteobacteria inf inf 0.000505306 3.41E−05 0k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 0.522843938 04.59E−05 4.54E−05 8.79E−05 f_Veillonellaceae; g_Veillonella k_Bacteria;p_Actinobacteria 2.88572859 3.028147004 0.010686058 0.007453190.003703071 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;5.713487631 6.369176097 0.018777814 0.015906199 0.003286576 f_(—)k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 5.7134876316.369176097 0.018777814 0.015906199 0.003286576 f_; g_(—) k_Bacteria;p_Actinobacteria; c_Actinobacteria; o_Bifidobacteriales 3.8076752383.673005325 0.010264517 0.007225959 0.002695744 k_Bacteria;p_Actinobacteria; c_Actinobacteria; o_Bifidobacteriales; 3.8076752383.673005325 0.010264517 0.007225959 0.002695744 f_Bifidobacteriaceaek_Bacteria; p_Actinobacteria; c_Actinobacteria; o_Bifidobacteriales;3.807675238 3.664672999 0.010264517 0.007225959 0.002695744f_Bifidobacteriaceae; g_Bifidobacterium k_Bacteria; p_Actinobacteria;c_Actinobacteria 3.742668409 3.626520891 0.010264517 0.0072714050.002742566 k_Bacteria; p_Actinobacteria; c_Coriobacteriia;o_Coriobacteriales; 0 0.585844581 0 0 9.42E−05 f_Coriobacteriaceae;g_Eggerthella k_Bacteria; p_Bacteroidetes 0.344495799 0.8261389170.210193161 0.404768129 0.610147241 k_Bacteria; p_Bacteroidetes;c_Bacteroidia 0.344418317 0.825842598 0.21011159 0.404637424 0.610047663k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales 0.3444183170.825842598 0.21011159 0.404637424 0.610047663 k_Bacteria; p_Firmicutes;Other; Other; Other; Other 1.03418292 2.13255533 0.000147539 0.00021050.000142663 k_Bacteria; p_Firmicutes; Other; Other 1.03418292 2.132555330.000147539 0.0002105 0.000142663 k_Bacteria; p_Firmicutes; Other;Other; Other 1.03418292 2.13255533 0.000147539 0.0002105 0.000142663k_Bacteria; p_Firmicutes; Other 1.03418292 2.13255533 0.0001475390.0002105 0.000142663 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 1.747310327 2.016617709 0.007878736 0.0088879590.004509065 f_Ruminococcaceae; Other k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.247844155 0.709404861 1.63E−05 3.36E−056.58E−05 f_Ruminococcaceae; g_Anaerotruncus k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.268505262 0.630391858 0.1270066560.248714588 0.473013658 f_Bacteroidaceae k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.268505262 0.630340467 0.1270066560.248714588 0.473013658 f_Bacteroidaceae; g_Bacteroides k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 17.65719148 248.12200240.000948467 0.000221251 5.37E−05 f_Prevotellaceae k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 17.65719148 247.99235870.000948467 0.000221251 5.37E−05 f_Prevotellaceae; g_Prevotellak_Bacteria; p_Proteobacteria 2.192234341 2.272050415 0.0382512510.01496568 0.017448523 k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.433529741 2.062863986 0.009768805 0.0263763270.022533183 f_Porphyromonadaceae; g_Parabacteroides k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.411630428 1.9592366160.009768805 0.026376327 0.02373198 f_Porphyromonadaceae k_Bacteria;p_Firmicutes; c_Clostridia; Other; Other; inf inf 0 0 0 Otherk_Bacteria; p_Firmicutes; c_Clostridia; Other inf inf 0 0 0 k_Bacteria;p_Firmicutes; c_Clostridia; Other; Other inf inf 0 0 0 k_Bacteria;p_Actinobacteria; c_Coriobacteriia; o_Coriobacteriales; 0.4267570487.362725286 3.26E−05 0 7.65E−05 f_Coriobacteriaceae; g_Collinsellak_Bacteria; p_Actinobacteria; c_Coriobacteriia; o_Coriobacteriales;0.517161809 1.879491712 0.0003426 0.000522821 0.000662462f_Coriobacteriaceae k_Bacteria; p_Actinobacteria; c_Coriobacteriia0.517161809 1.879491712 0.0003426 0.000522821 0.000662462 k_Bacteria;p_Actinobacteria; c_Coriobacteriia; o_Coriobacteriales 0.5171618091.879491712 0.0003426 0.000522821 0.000662462 k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 3.017549229 0.696152119 0.0361687550.013559835 0.011986136 f_Lachnospiraceae; Other k_Bacteria; Other;Other; Other 1.301001458 1.570025829 0.000513831 0.000716455 0.00039495k_Bacteria; Other; Other; Other; Other; Other 1.301001458 1.5700258290.000513831 0.000716455 0.00039495 k_Bacteria; Other; Other 1.3010014581.570025829 0.000513831 0.000716455 0.00039495 k_Bacteria; Other; Other;Other; Other 1.301001458 1.570025829 0.000513831 0.000716455 0.00039495k_Bacteria; Other 1.301001458 1.570025829 0.000513831 0.0007164550.00039495 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;2.683981412 0 0.000126462 0 4.71E−05 f_Lachnospiraceae;g_Pseudobutyrivibrio k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.058011525 0.514869524 0.002008032 0.0157443490.034614365 f_Rikenellaceae; g_Alistipes k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 1.276736899 1.974407509 0.0005427110.000836392 0.000425077 f_Lachnospiraceae; g_Anaerostipes k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.524902192 1.4178237140.01100073 0.020311601 0.020957675 f_Ruminococcaceae; g_Oscillospirak_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 5.4551294352.094743099 0.002339551 0.000637078 0.000428872 f_Ruminococcaceae;g_Butyricicoccus k_Bacteria; p_Actinobacteria; c_Actinobacteria;o_Actinomycetales; 0 0 0 0 3.15E−05 f_Actinomycetaceae; g_Actinomycesk_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.1201107172.207642958 0.000967321 0.009714929 0.008053581 f_Rikenellaceae; g_(—)k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales 0.7699665670.649684935 0.00048844 0.000793279 0.000634366 k_Bacteria;p_Proteobacteria; c_Gammaproteobacteria; 0 0 0 0 9.56E−06o_Enterobacteriales; f_Enterobacteriaceae; g_Klebsiella k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.994459647 1.5991428493.26E−05 1.88E−05 3.28E−05 f_[Tissierellaceae] k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 1.453704892 1.110880946 0.2305572140.230537983 0.158599738 f_Ruminococcaceae k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.831155585 4.796755878 0.0011776090.001908744 0.001416833 f_[Odoribacteraceae] k_Bacteria; p_Firmicutes;c_Bacilli; o_Lactobacillales; 0.699563524 0.706905977 0.0004078570.000242931 0.000583016 f_Streptococcaceae k_Bacteria; p_Proteobacteria;c_Gammaproteobacteria; 1.193837765 0 4.59E−05 0 3.85E−05o_Enterobacteriales; f_Enterobacteriaceae; g_Shigella k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 5.11663948 1.4401521270.021681676 0.005215694 0.004237484 f_Lachnospiraceae; g_Lachnospirak_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 2.2019818080.70751989 0.01978803 0.012243708 0.008986464 f_Lachnospiraceae;g_Clostridium k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;3.523547829 3.422413343 0.003709558 0.001636066 0.001052791f_Veillonellaceae; g_Phascolarctobacterium k_Bacteria; p_Firmicutes;c_Bacilli; o_Lactobacillales; 0.590616304 0.722103841 0.0003367570.000242931 0.000570179 f_Streptococcaceae; g_Streptococcus k_Bacteria;p_Proteobacteria; c_Gammaproteobacteria; 6.817633863 0.3373944520.000954145 6.29E−05 0.000139953 o_Pasteurellales; f_Pasteurellaceae;g_Haemophilus k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;3.357260558 1.594334641 0.012807989 0.00340103 0.003815012f_Peptostreptococcaceae k_Bacteria; p_Proteobacteria;c_Gammaproteobacteria; 7.00622958 0.430120039 0.00107048 0.000106180.00015279 o_Pasteurellales k_Bacteria; p_Proteobacteria;c_Gammaproteobacteria; 7.00622958 0.430120039 0.00107048 0.000106180.00015279 o_Pasteurellales; f_Pasteurellaceae k_Bacteria; p_Firmicutes;c_Bacilli 1.467041864 0.940659447 0.00162713 0.001510384 0.001109123k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 1.8131069280.790901395 0.282307099 0.178019629 0.155703503 f_Lachnospiraceaek_Bacteria; p_Bacteroidetes; Other; Other; Other; Other 0.5161761781.201854876 8.16E−05 7.56E−05 0.00015803 k_Bacteria; p_Bacteroidetes;Other; Other 0.516176178 1.201854876 8.16E−05 7.56E−05 0.00015803k_Bacteria; p_Bacteroidetes; Other 0.516176178 1.201854876 8.16E−057.56E−05 0.00015803 k_Bacteria; p_Bacteroidetes; Other; Other; Other0.516176178 1.201854876 8.16E−05 7.56E−05 0.00015803 k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.981525478 0.909490210.002480592 0.003488372 0.002527282 Other k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.981525478 0.90949021 0.0024805920.003488372 0.002527282 Other; Other k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 1.850680871 0.812111071 0.0353883440.020691033 0.019121797 f_Lachnospiraceae; g_Coprococcus k_Bacteria;p_Verrucomicrobia; c_Verrucomicrobiae; 0.257961535 0.7288983330.000551243 0.004473737 0.002136918 o_Verrucomicrobiales;f_Verrucomicrobiaceae; g_Akkermansia k_Bacteria; p_Verrucomicrobia;c_Verrucomicrobiae; 0.254058759 0.717870614 0.000551243 0.0044737370.002169744 o_Verrucomicrobiales k_Bacteria; p_Verrucomicrobia;c_Verrucomicrobiae; 0.254058759 0.717870614 0.000551243 0.0044737370.002169744 o_Verrucomicrobiales; f_Verrucomicrobiaceae k_Bacteria;p_Verrucomicrobia 0.254058759 0.717870614 0.000551243 0.0044737370.002169744 k_Bacteria; p_Verrucomicrobia; c_Verrucomicrobiae0.254058759 0.717870614 0.000551243 0.004473737 0.002169744 k_Bacteria;p_Tenericutes inf inf 0.000880971 0.000499909 0 k_Bacteria;p_Firmicutes; c_Clostridia 1.625088942 1.142513703 0.5150443290.496217231 0.31693301 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales 1.625088942 1.142513703 0.515044329 0.4959239930.31693301 k_Bacteria; p_Firmicutes; c_Erysipelotrichi;o_Erysipelotrichales; 1.269226262 0.43813951 0.0056043 0.0053089830.004415525 f_Erysipelotrichaceae; g_(—) k_Bacteria; p_Actinobacteria;c_Coriobacteriia; o_Coriobacteriales; 1.243790419 1.2883749970.000105144 9.09E−05 8.45E−05 f_Coriobacteriaceae; g_(—) k_Bacteria;p_Firmicutes 1.601955527 1.167896058 0.521797051 0.523011392 0.325725054k_Bacteria; p_Proteobacteria; c_Betaproteobacteria 3.2653114621.561772748 0.023853623 0.008376604 0.00730516 k_Bacteria;p_Proteobacteria; c_Betaproteobacteria; o_Burkholderiales 3.2653114621.557543063 0.023853623 0.008376604 0.00730516 k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 9.072677791 0.974782185 0.00079949.09E−05 8.81E−05 f_Lachnospiraceae; g_Lachnobacterium k_Bacteria;p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales 0.9085614860.469731563 0.006201479 0.007736019 0.006825602 k_Bacteria;p_Firmicutes; c_Erysipelotrichi 0.908561486 0.469731563 0.0062014790.007736019 0.006825602 k_Bacteria; p_Firmicutes; c_Erysipelotrichi;o_Erysipelotrichales; 0.908561486 0.469731563 0.006201479 0.0077360190.006825602 f_Erysipelotrichaceae k_Bacteria; p_Proteobacteria;c_Betaproteobacteria; o_Burkholderiales; 3.148210789 1.4725943740.02228209 0.008376604 0.0070777 f_Alcaligenaceae k_Bacteria;p_Proteobacteria; c_Betaproteobacteria; o_Burkholderiales; 3.1402807871.464825274 0.022225964 0.008376604 0.0070777 f_Alcaligenaceae;g_Sutterella k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;1.306385665 1.029190844 0.025648711 0.021260472 0.019633338f_Lachnospiraceae; g_Blautia k_Bacteria; p_Firmicutes;c_Erysipelotrichi; o_Erysipelotrichales; 0 0 0 6.29E−05 4.53E−05f_Erysipelotrichaceae; g_Coprobacillus k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.536601183 1.743188226 0.00011420.000221251 0.000212821 Other k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.536601183 1.743188226 0.00011420.000221251 0.000212821 Other; Other k_Bacteria; p_Actinobacteria;c_Actinobacteria; o_Actinomycetales 0 1.218333024 0 3.75E−05 4.61E−05k_Bacteria; p_Proteobacteria; c_Deltaproteobacteria; 0.8697942691.174806104 0.001031152 0.000932554 0.001185512 o_Desulfovibrionales;f_Desulfovibrionaceae; g_Bilophila k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 2.084966197 1.007207434 0.0047001790.001875469 0.002254319 f_Peptostreptococcaceae; Other k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 1.518402522 0.7974785680.005661055 0.003284265 0.003728296 f_Clostridiaceae; g_Clostridiumk_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 1.0841329341.272777939 0.003169645 0.004715194 0.002923668 f_Lachnospiraceae;g_Dorea k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;3.022482575 1.27215778 0.157954842 0.071420012 0.052259968f_Ruminococcaceae; g_Faecalibacterium k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 1.048841882 1.267345199 0.0002773430.000252042 0.000264428 f_Peptostreptococcaceae; g_Clostridiumk_Bacteria; p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales;0.621776194 0.823944028 0.000309971 0.000388689 0.000498525f_Erysipelotrichaceae; g_Clostridium k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 2.504352946 13.14941079 4.21E−050.000971723 1.68E−05 f_Clostridiaceae; g_(—) k_Bacteria; p_Firmicutes;c_Erysipelotrichi; o_Erysipelotrichales; 2.309460668 1.4280204680.000183748 9.29E−05 7.96E−05 f_Erysipelotrichaceae; g_Holdemaniak_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 1.3621449750.610778314 0.008692301 0.006038584 0.006381333 f_Clostridiaceaek_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 0 0.901570882 06.29E−05 8.79E−05 f_Ruminococcaceae; g_Clostridium k_Bacteria;p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; 0 0.419550281 00.000212359 0.000157073 f_Erysipelotrichaceae; g_[Eubacterium]k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 2.5833869220.717930175 0.074168314 0.042863063 0.02870972 f_Lachnospiraceae;g_Roseburia k_Bacteria; p_Actinobacteria; c_Actinobacteria;o_Actinomycetales; 0 0.647292947 0 0 3.47E−05 f_Actinomycetaceaek_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 3.2967071940.766585538 0.000954145 0.000201633 0.000289424 f_Clostridiaceae;g_SMB53 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;1.00601522 1.062751999 0.007882812 0.007641034 0.007835678f_Lachnospiraceae; g_[Ruminococcus] k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.536877265 1.443733623 0.0002756210.001406267 0.000513379 f_Christensenellaceae; g_(—) k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 2.670911283 0.6836346560.033267443 0.014794342 0.012455465 f_Lachnospiraceae; g_(—) k_Bacteria;p_Proteobacteria; c_Gammaproteobacteria; 1.022906515 1.9308065540.000505136 0.000243811 0.000493824 o_Enterobacteriales;f_Enterobacteriaceae; g_Escherichia k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 1.137356676 1.741723987 0.000556650.001437758 0.000489424 f_[Mogibacteriaceae]; g_(—) k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.536877265 1.5182565480.000275621 0.001406267 0.000513379 f_Christensenellaceae k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 1.472940024 1.514854430.028288958 0.0437382 0.019205778 f_Ruminococcaceae; g_Ruminococcusk_Bacteria; p_Firmicutes; c_Bacilli; o_Turicibacterales; 1.2159356960.955712453 0.000299735 0.000145758 0.000246505 f_Turicibacteraceae;g_Turicibacter k_Bacteria; p_Firmicutes; c_Bacilli; o_Turicibacterales;1.215935696 0.955712453 0.000299735 0.000145758 0.000246505f_Turicibacteraceae k_Bacteria; p_Firmicutes; c_Bacilli;o_Turicibacterales 1.215935696 0.955712453 0.000299735 0.0001457580.000246505 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;87.44384578 1.70820069 0.023443618 0.00337408 0.000268099f_Veillonellaceae; g_Dialister k_Bacteria; p_Proteobacteria;c_Gammaproteobacteria; 2.302029237 1.663637994 0.001682405 0.00033680.000730836 o_Enterobacteriales; f_Enterobacteriaceae k_Bacteria;p_Proteobacteria; c_Gammaproteobacteria; 2.302029237 1.6636379940.001682405 0.0003368 0.000730836 o_Enterobacteriales k_Bacteria;p_Proteobacteria; c_Gammaproteobacteria; 0.509840777 1.3913309325.43E−05 7.21E−05 0.000106447 o_Enterobacteriales; f_Enterobacteriaceae;Other k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales;0.147361573 1.109505141 0.00033646 0.001516181 0.002283225f_Rikenellaceae; Other k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 9.667924155 25.23844637 0.000379898 0.0075868423.93E−05 f_[Barnesiellaceae] k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 9.667924155 25.23844637 0.000379898 0.0071533083.93E−05 f_[Barnesiellaceae]; g_(—) k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 1.137356676 1.741723987 0.000556650.001437758 0.000489424 f_[Mogibacteriaceae] k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 4.806417425 0.984253727 0.0303375660.00905059 0.006311888 f_Veillonellaceae k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 1.068251023 1.746723821 0.0010724650.001052221 0.001003945 f_[Odoribacteraceae]; g_Odoribacter k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.060561698 0.7895824810.00330151 0.03397749 0.054514817 f_Rikenellaceae k_Bacteria;p_Proteobacteria; c_Gammaproteobacteria 1.745513701 1.3020223070.002229045 0.000759536 0.001277014 k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.708114468 0.856367273 0.0107032940.031857844 0.015115203 f_Ruminococcaceae; g_(—) Final ASD FDR- mediancorrected Taxonomy abundance Wilcoxon p-value p-value (q) k_Bacteria;p_Proteobacteria; c_Betaproteobacteria; o_Burkholderiales; 0.000142986 06.32E−05 0.00273954 f_Oxalobacteraceae k_Bacteria; p_Proteobacteria;c_Betaproteobacteria; o_Burkholderiales; 0.000116622 0 7.90E−050.00273954 f_Oxalobacteraceae; g_Oxalobacter k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.000957645 3 8.72E−050.00273954 f_S24-7; g_(—) k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.000957645 3 8.72E−05 0.00273954 f_S24-7 k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.018320426 5 6.48E−050.00273954 f_Ruminococcaceae; g_Gemmiger k_Bacteria; p_Proteobacteria;c_Betaproteobacteria; o_Burkholderiales; 5.84E−05 13 0.00032 0.00840514f_Comamonadaceae k_Bacteria; p_Proteobacteria; c_Deltaproteobacteria;0.006564225 14 0.00053 0.01179367 o_Desulfovibrionales;f_Desulfovibrionaceae; g_Desulfovibrio k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 4.71E−05 19 0.00238 0.02249607f_Ruminococcaceae; g_Anaerofilum k_Bacteria; p_Proteobacteria;c_Betaproteobacteria; o_Burkholderiales; 5.62E−05 20 0.00115 0.02011689Other; Other k_Bacteria; p_Proteobacteria; c_Betaproteobacteria;o_Burkholderiales; 5.62E−05 20 0.00115 0.02011689 Other k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 5.58E−05 22 0.002970.02249607 f_Ruminococcaceae; g_Subdoligranulum k_Bacteria;p_Proteobacteria; c_Alphaproteobacteria; 0.001304964 23 0.002950.02249607 o_RF32 k_Bacteria; p_Proteobacteria; c_Alphaproteobacteria;0.001304964 23 0.00295 0.02249607 o_RF32; f_; g_(—) k_Bacteria;p_Proteobacteria; c_Alphaproteobacteria; 0.001304964 23 0.002950.02249607 o_RF32; f_(—) k_Bacteria; p_Lentisphaerae; c_[Lentisphaeria];o_Victivallales 3.00E−05 24 0.00228 0.02249607 k_Bacteria;p_Lentisphaerae; c_[Lentisphaeria] 3.00E−05 24 0.00228 0.02249607k_Bacteria; p_Lentisphaerae 3.00E−05 24 0.00228 0.02249607 k_Bacteria;p_Lentisphaerae; c_[Lentisphaeria]; o_Victivallales; 3.00E−05 24 0.002280.02249607 f_Victivallaceae k_Bacteria; p_Actinobacteria;c_Coriobacteriia; o_Coriobacteriales; 0.000199396 26 0.00315 0.02249607f_Coriobacteriaceae; g_Adlercreutzia k_Bacteria; p_Proteobacteria;c_Deltaproteobacteria; 0.008161503 26 0.00315 0.02249607o_Desulfovibrionales; f_Desulfovibrionaceae k_Bacteria;p_Proteobacteria; c_Deltaproteobacteria; 0.008161503 26 0.003150.02249607 o_Desulfovibrionales k_Bacteria; p_Proteobacteria;c_Deltaproteobacteria 0.008161503 26 0.00315 0.02249607 k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.004541428 28 0.004330.02954072 f_[Odoribacteraceae]; g_Butyricimonas k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0 28 0.00625 0.03922132f_Lachnospiraceae; g_Ruminococcus k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.002480787 29 0.00505 0.033057f_Peptostreptococcaceae; g_(—) k_Bacteria; p_Proteobacteria;c_Alphaproteobacteria 0.001329582 30 0.0083 0.04495085 k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0 30 0.01124 0.0534674f_Veillonellaceae; g_Veillonella k_Bacteria; p_Actinobacteria0.011213443 31 0.00684 0.04132322 k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.020932784 32 0.00794 0.04450209 f_(—)k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 0.020932784 320.00794 0.04450209 f_; g_(—) k_Bacteria; p_Actinobacteria;c_Actinobacteria; o_Bifidobacteriales 0.009901481 32 0.01095 0.0534674k_Bacteria; p_Actinobacteria; c_Actinobacteria; o_Bifidobacteriales;0.009901481 32 0.01095 0.0534674 f_Bifidobacteriaceae k_Bacteria;p_Actinobacteria; c_Actinobacteria; o_Bifidobacteriales; 0.009879019 320.01095 0.0534674 f_Bifidobacteriaceae; g_Bifidobacterium k_Bacteria;p_Actinobacteria; c_Actinobacteria 0.009945975 35 0.01221 0.05640206k_Bacteria; p_Actinobacteria; c_Coriobacteriia; o_Coriobacteriales;5.52E−05 36 0.02229 0.07523254 f_Coriobacteriaceae; g_Eggerthellak_Bacteria; p_Bacteroidetes 0.50406638 37 0.0161 0.06831558 k_Bacteria;p_Bacteroidetes; c_Bacteroidia 0.503803347 37 0.0161 0.06831558k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales 0.50380334737 0.0161 0.06831558 k_Bacteria; p_Firmicutes; Other; Other; Other;Other 0.000304236 38 0.01842 0.06886366 k_Bacteria; p_Firmicutes; Other;Other 0.000304236 38 0.01842 0.06886366 k_Bacteria; p_Firmicutes; Other;Other; Other 0.000304236 38 0.01842 0.06886366 k_Bacteria; p_Firmicutes;Other 0.000304236 38 0.01842 0.06886366 k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.00909306 38 0.01842 0.06886366f_Ruminococcaceae; Other k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 4.67E−05 39 0.027 0.08312852 f_Ruminococcaceae;g_Anaerotruncus k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.298183959 39 0.02103 0.07504768 f_Bacteroidaceaek_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.2981596539 0.02103 0.07504768 f_Bacteroidaceae; g_Bacteroides k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.013328022 40 0.023960.07523254 f_Prevotellaceae k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.013321059 40 0.02396 0.07523254 f_Prevotellaceae;g_Prevotella k_Bacteria; p_Proteobacteria 0.039643924 40 0.023960.07523254 k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales;0.046482891 40 0.02396 0.07523254 f_Porphyromonadaceae;g_Parabacteroides k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.046496563 40 0.02396 0.07523254 f_Porphyromonadaceaek_Bacteria; p_Firmicutes; c_Clostridia; Other; Other; 3.28E−05 41 0.0450.10868376 Other k_Bacteria; p_Firmicutes; c_Clostridia; Other 3.28E−0541 0.045 0.10868376 k_Bacteria; p_Firmicutes; c_Clostridia; Other; Other3.28E−05 41 0.045 0.10868376 k_Bacteria; p_Actinobacteria;c_Coriobacteriia; o_Coriobacteriales; 0.000562932 41 0.03992 0.10275249f_Coriobacteriaceae; g_Collinsella k_Bacteria; p_Actinobacteria;c_Coriobacteriia; o_Coriobacteriales; 0.001245091 42 0.03089 0.08979861f_Coriobacteriaceae k_Bacteria; p_Actinobacteria; c_Coriobacteriia0.001245091 42 0.03089 0.08979861 k_Bacteria; p_Actinobacteria;c_Coriobacteriia; o_Coriobacteriales 0.001245091 42 0.03089 0.08979861k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 0.008344174 430.03495 0.09977185 f_Lachnospiraceae; Other k_Bacteria; Other; Other;Other 0.000620082 44 0.03947 0.10275249 k_Bacteria; Other; Other; Other;Other; Other 0.000620082 44 0.03947 0.10275249 k_Bacteria; Other; Other0.000620082 44 0.03947 0.10275249 k_Bacteria; Other; Other; Other; Other0.000620082 44 0.03947 0.10275249 k_Bacteria; Other 0.000620082 440.03947 0.10275249 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 0 44 0.06007 0.13667366 f_Lachnospiraceae;g_Pseudobutyrivibrio k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.017821882 45 0.04447 0.10868376 f_Rikenellaceae;g_Alistipes k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;0.000839275 46 0.04999 0.11714052 f_Lachnospiraceae; g_Anaerostipesk_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 0.029714289 460.04999 0.11714052 f_Ruminococcaceae; g_Oscillospira k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.000898376 47 0.056080.12947425 f_Ruminococcaceae; g_Butyricicoccus k_Bacteria;p_Actinobacteria; c_Actinobacteria; o_Actinomycetales; 0 47 0.075480.16459142 f_Actinomycetaceae; g_Actinomyces k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.017779431 48 0.062770.13880434 f_Rikenellaceae; g_(—) k_Bacteria; p_Firmicutes; c_Bacilli;o_Lactobacillales 0.000412138 48 0.06277 0.13880434 k_Bacteria;p_Proteobacteria; c_Gammaproteobacteria; 0 48 0.08672 0.18192579o_Enterobacteriales; f_Enterobacteriaceae; g_Klebsiella k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 5.25E−05 49 0.076730.16501965 f_[Tissierellaceae] k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 0.176185427 51 0.08691 0.18192579 f_Ruminococcaceaek_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.00679620252 0.09645 0.19924207 f_[Odoribacteraceae] k_Bacteria; p_Firmicutes;c_Bacilli; o_Lactobacillales; 0.000412138 53 0.10681 0.21778096f_Streptococcaceae k_Bacteria; p_Proteobacteria; c_Gammaproteobacteria;0 53 0.14088 0.2764745 o_Enterobacteriales; f_Enterobacteriaceae;g_Shigella k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;0.006102621 54 0.11803 0.23457433 f_Lachnospiraceae; g_Lachnospirak_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 0.006358102 540.11803 0.23457433 f_Lachnospiraceae; g_Clostridium k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.003603085 57 0.15730.29754187 f_Veillonellaceae; g_Phascolarctobacterium k_Bacteria;p_Firmicutes; c_Bacilli; o_Lactobacillales; 0.000411728 57 0.15730.29754187 f_Streptococcaceae; g_Streptococcus k_Bacteria;p_Proteobacteria; c_Gammaproteobacteria; 4.72E−05 57 0.1573 0.29754187o_Pasteurellales; f_Pasteurellaceae; g_Haemophilus k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.006082406 58 0.172380.31469611 f_Peptostreptococcaceae k_Bacteria; p_Proteobacteria;c_Gammaproteobacteria; 6.57E−05 58 0.17238 0.31469611 o_Pasteurellalesk_Bacteria; p_Proteobacteria; c_Gammaproteobacteria; 6.57E−05 58 0.172380.31469611 o_Pasteurellales; f_Pasteurellaceae k_Bacteria; p_Firmicutes;c_Bacilli 0.001043307 59 0.18852 0.34020243 k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.123146118 60 0.20575 0.36707003f_Lachnospiraceae k_Bacteria; p_Bacteroidetes; Other; Other; Other;Other 0.000189929 61 0.2348 0.39217229 k_Bacteria; p_Bacteroidetes;Other; Other 0.000189929 61 0.2348 0.39217229 k_Bacteria;p_Bacteroidetes; Other 0.000189929 61 0.2348 0.39217229 k_Bacteria;p_Bacteroidetes; Other; Other; Other 0.000189929 61 0.2348 0.39217229k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 0.002298538 610.22409 0.3909104 Other k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 0.002298538 61 0.22409 0.3909104 Other; Otherk_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 0.015529023 630.26421 0.43211663 f_Lachnospiraceae; g_Coprococcus k_Bacteria;p_Verrucomicrobia; c_Verrucomicrobiae; 0.001557596 63 0.27523 0.43211663o_Verrucomicrobiales; f_Verrucomicrobiaceae; g_Akkermansia k_Bacteria;p_Verrucomicrobia; c_Verrucomicrobiae; 0.001557596 63 0.27523 0.43211663o_Verrucomicrobiales k_Bacteria; p_Verrucomicrobia; c_Verrucomicrobiae;0.001557596 63 0.27523 0.43211663 o_Verrucomicrobiales;f_Verrucomicrobiaceae k_Bacteria; p_Verrucomicrobia 0.001557596 630.27523 0.43211663 k_Bacteria; p_Verrucomicrobia; c_Verrucomicrobiae0.001557596 63 0.27523 0.43211663 k_Bacteria; p_Tenericutes 0.00012624764 0.32999 0.49815875 k_Bacteria; p_Firmicutes; c_Clostridia 0.36210030664 0.28603 0.43599322 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales 0.362100306 64 0.28603 0.43599322 k_Bacteria;p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales; 0.001934616 640.28603 0.43599322 f_Erysipelotrichaceae; g_(—) k_Bacteria;p_Actinobacteria; c_Coriobacteriia; o_Coriobacteriales; 0.000108913 660.34435 0.51488091 f_Coriobacteriaceae; g_(—) k_Bacteria; p_Firmicutes0.380413007 67 0.35862 0.53116324 k_Bacteria; p_Proteobacteria;c_Betaproteobacteria 0.011409 68 0.38519 0.54481563 k_Bacteria;p_Proteobacteria; c_Betaproteobacteria; o_Burkholderiales 0.011378101 680.38519 0.54481563 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 8.59E−05 68 0.41288 0.56373387 f_Lachnospiraceae;g_Lachnobacterium k_Bacteria; p_Firmicutes; c_Erysipelotrichi;o_Erysipelotrichales 0.003206201 68 0.38519 0.54481563 k_Bacteria;p_Firmicutes; c_Erysipelotrichi 0.003206201 68 0.38519 0.54481563k_Bacteria; p_Firmicutes; c_Erysipelotrichi; o_Erysipelotrichales;0.003206201 68 0.38519 0.54481563 f_Erysipelotrichaceae k_Bacteria;p_Proteobacteria; c_Betaproteobacteria; o_Burkholderiales; 0.01042258169 0.41293 0.56373387 f_Alcaligenaceae k_Bacteria; p_Proteobacteria;c_Betaproteobacteria; o_Burkholderiales; 0.010367593 69 0.412930.56373387 f_Alcaligenaceae; g_Sutterella k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.020206452 69 0.41293 0.56373387f_Lachnospiraceae; g_Blautia k_Bacteria; p_Firmicutes;c_Erysipelotrichi; o_Erysipelotrichales; 0 69 0.45471 0.58515603f_Erysipelotrichaceae; g_Coprobacillus k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.000370987 70 0.44181 0.57326273 Otherk_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.00037098770 0.44181 0.57326273 Other; Other k_Bacteria; p_Actinobacteria;c_Actinobacteria; o_Actinomycetales 5.62E−05 70 0.44181 0.57326273k_Bacteria; p_Proteobacteria; c_Deltaproteobacteria; 0.001392747 700.44181 0.57326273 o_Desulfovibrionales; f_Desulfovibrionaceae;g_Bilophila k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;0.002270567 70 0.44181 0.57326273 f_Peptostreptococcaceae; Otherk_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 0.002973236 700.44181 0.57326273 f_Clostridiaceae; g_Clostridium k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.00372118 71 0.471830.59261272 f_Lachnospiraceae; g_Dorea k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.066482925 71 0.47183 0.59261272f_Ruminococcaceae; g_Faecalibacterium k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.000335121 71 0.47183 0.59261272f_Peptostreptococcaceae; g_Clostridium k_Bacteria; p_Firmicutes;c_Erysipelotrichi; o_Erysipelotrichales; 0.000410757 71 0.481960.60054299 f_Erysipelotrichaceae; g_Clostridium k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.000220828 72 0.52760.64211601 f_Clostridiaceae; g_(—) k_Bacteria; p_Firmicutes;c_Erysipelotrichi; o_Erysipelotrichales; 0.000113618 72 0.512690.62884726 f_Erysipelotrichaceae; g_Holdemania k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.00389758 72 0.50293 0.62172928f_Clostridiaceae k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 7.92E−05 73 0.55219 0.66178605 f_Ruminococcaceae;g_Clostridium k_Bacteria; p_Firmicutes; c_Erysipelotrichi;o_Erysipelotrichales; 6.59E−05 73 0.53508 0.64621276f_Erysipelotrichaceae; g_[Eubacterium] k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.020611574 74 0.56824 0.6758595f_Lachnospiraceae; g_Roseburia k_Bacteria; p_Actinobacteria;c_Actinobacteria; o_Actinomycetales; 2.25E−05 74 0.59051 0.69707032f_Actinomycetaceae k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 0.000221868 75 0.60235 0.70573682 f_Clostridiaceae;g_SMB53 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;0.008327383 76 0.63735 0.74121664 f_Lachnospiraceae; g_[Ruminococcus]k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 0.000741182 780.70977 0.81936988 f_Christensenellaceae; g_(—) k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.008514988 79 0.747040.8557764 f_Lachnospiraceae; g_(—) k_Bacteria; p_Proteobacteria;c_Gammaproteobacteria; 0.000953478 80 0.78492 0.8557764o_Enterobacteriales; f_Enterobacteriaceae; g_Escherichia k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.000852442 80 0.784920.8557764 f_[Mogibacteriaceae]; g_(—) k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; 0.00077944 80 0.78492 0.8557764f_Christensenellaceae k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; 0.029093957 80 0.78492 0.8557764 f_Ruminococcaceae;g_Ruminococcus k_Bacteria; p_Firmicutes; c_Bacilli; o_Turicibacterales;0.000235588 80 0.78492 0.8557764 f_Turicibacteraceae; g_Turicibacterk_Bacteria; p_Firmicutes; c_Bacilli; o_Turicibacterales; 0.000235588 800.78492 0.8557764 f_Turicibacteraceae k_Bacteria; p_Firmicutes;c_Bacilli; o_Turicibacterales 0.000235588 80 0.78492 0.8557764k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; 0.000457967 820.86212 0.90840897 f_Veillonellaceae; g_Dialister k_Bacteria;p_Proteobacteria; c_Gammaproteobacteria; 0.001215846 82 0.862120.90840897 o_Enterobacteriales; f_Enterobacteriaceae k_Bacteria;p_Proteobacteria; c_Gammaproteobacteria; 0.001215846 82 0.862120.90840897 o_Enterobacteriales k_Bacteria; p_Proteobacteria;c_Gammaproteobacteria; 0.000148103 82 0.86212 0.90840897o_Enterobacteriales; f_Enterobacteriaceae; Other k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.00253325 82 0.862120.90840897 f_Rikenellaceae; Other k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.000991737 83 0.90127 0.92483534f_[Barnesiellaceae] k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.000991737 83 0.90127 0.92483534 f_[Barnesiellaceae];g_(—) k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;0.000852442 83 0.90127 0.92483534 f_[Mogibacteriaceae] k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; 0.006212499 83 0.901270.92483534 f_Veillonellaceae k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.001753614 84 0.94067 0.95280405 f_[Odoribacteraceae];g_Odoribacter k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.043043944 84 0.94067 0.95280405 f_Rikenellaceaek_Bacteria; p_Proteobacteria; c_Gammaproteobacteria 0.0016627 85 0.980210.98020591 k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;0.012944165 85 0.98021 0.98020591 f_Ruminococcaceae; g_(—)

Example 14: Microbiome Analysis Across Common Diversity Metrics

Parallel analyses are performed for multiple diversity metrics tounderstand the effect of metric on the microbiome findings. With theObserved OTUs metric (a count of the number of OTUs observed at leasttwo times in a sample (FIG. 16), the same patterns as in FIG. 14, panela are observed. This confirms that both phylogenetic andnon-phylogenetic metrics illustrate the same patterns of change incommunity richness with MTT.

Similarly, pairwise distances are computed between samples using fourdiversity metrics, a qualitative non-phylogenetic metric (Jaccarddistance), and quantitative non-phylogenetic diversity metric(Bray-Curtis distance), a qualitative phylogenetic diversity metric(unweighted UniFrac), and a quantitative phylogenetic diversity metric(weighted UniFrac) (FIG. 17). While the same pattern is observed withthe first three metrics, no significant engraftment is seen withweighted UniFrac (though less variation is observed across individualsat weeks 10 and 18 than in earlier timepoints). Quantitative metricsgive more weight to higher abundance OTUs than qualitative metrics.Because differences are observed in composition with our quantitativenon-phylogenetic metric but not our quantitative phylogenetic metric,without being bound to any scientific theory, this may suggest that whenchanges occur in high abundance OTUs, those OTUs are generally closelyrelated (thus the change is down-weighted with a phylogenetic diversitymetric relative to a non-phylogenetic diversity metric).

Example 15: Microbiome Analysis Based on Fecal Swab Samples

In addition to stool collection over the 18 week period, fecal swabsamples are collected nearly every other week. These samples areobtained by swabbing bottoms or used toilet paper with a sterile swab,and thus are easier to collect than stool samples. The general patternsobserved from the stool data are present in the swab data. However, thefecal swab samples do not achieve the same statistical significance asin the stool samples (FIG. 18). This could be due to greater variabilityin the handling of the swab samples, which are shipped to our collectionfacility at ASU, and thus spent a varied amount of time at ambienttemperature, ranging from a few hours to multiple days.

Example 16: Bacteriophage Virome Analysis

Changes in the phage diversity of gut samples from ASD or neurotypicalparticipants are also evaluated. Briefly, phages are surveyed bysequencing community DNA from purified viral fraction samples,assembling them, and defining phage populations from the assembledcontigs as established in Brum et al., Patterns and ecological driversof ocean viral communities. Science 348, doi:10.1126/science.1261498(2015). Abundance profiles of phage populations are then statisticallyevaluated for changes over time.

In contrast to the microbiota, phage richness and evenness do notsignificantly change following MTT during the timeframe of this study(FIG. 19, panel a). Without being bound to any scientific theory, at thepopulation level, phage communities are reliant on their hostcommunities and, thus, significant changes in phage diversity can lagbehind bacterial community changes (Rodriguez-Brito et al. 2010).However, a number of metrics suggested phage communities also respondedto MTT as follows.

First, in four individuals tracked across all four stool samples, phagediversity of three MTT responders decrease and then recover, while thenon-responder decreases but does not recover. Second, analyses ofquantitative (Bray-Curtis; FIG. 19, panel b, right) and qualitative(Jaccard; FIG. 19, panel b, left) measures of community dissimilaritybetween samples revealed phage communities of ASD-afflicted children aresignificantly more similar to those from the donor following MTT.Permutation based fitting of subject variables to Bray-Curtis andJaccard NMDS plots uncovered significant clustering based on subjecttype (e.g. autism, neutrotypic and donor; r²≥0.2120, p-value ≤0.0001,9999 permutations) and among ASD subjects based on treatment stage(r²≥0.4021, p≤0.0002, 9999 permutations), and high (r²≥0.2066, p≤0.0149,9999 permutations) and low (r²≥0.1851, p≤0.0023, 9999 permutations) dosedonor. Not surprisingly, in addition to microbiota, FMT also transfersphages. Indeed, phage communities of ASD children appear driven by thesuccessful transfer of donor viral populations during MTT (FIG. 19,panel c). Following the high and low doses of MTT, phage populationsfrom the donor engrafted across all ASD subjects, while the abundance ofphage populations originally in their pre-MTT virome are completelyeliminated or decrease (FIG. 19, panel c). In contrast, acrossneurotypical subjects high abundance (>60%) of phage populations of thestarting virome remained indicating that the changes observed are notdue to normal temporal variability (FIG. 19, panel d). The resultsindicate that MTT shifts phage communities of ASD children towards thoseof donors

Procedures for gut virome preparation, sequence, and analysis aresummarized below. Viral DNA is isolated from stool samples as previouslydescribed by (Minot et al. Genome Research 21:1616-25 (2011)) withslight modifications. Briefly, 0.5 g of stool is resuspended into 40 mLof SM buffer and spun down at 4000 rpm for 30 min and the supernatant isfiltered at 0.2 um. The filtrate is then ultra-centrifuged in a stepCsCl gradient as detailed in (Thurber et al., Environmental Microbiology11:2148-63 (2009)). To target dsDNA bacteriophages, the 1.35-1.5 g/mLfraction is collected from the CsCl column, treated with chloroform(Vega Thurber et al 2009) and then with DNase I (100 U/mL) followed bythe addition of 0.1M EDTA and 0.1M EGTA to halt enzyme activity asdescribed (Hurwitz et al., Environmental Microbiology 15:1428-40(2013)). Viral DNA is then extracted using the DNeasy Blood and TissueKit.

Following DNA extraction, the NexteraXT kit is used to prepare thesequencing libraries with two minor changes. During the librarypreparation, input DNA is PCR amplified with 18-25 cycles. When inputDNA concentrations are low, the buffer ATM is added at a 1:10 dilution.Sequencing is performed on a MiSeq v3 2×300 at one-sixth of a lane persequencing library.

For Virome bioinformatic analysis, reads are QC′d using Trimmomatic(Bolger et al., Bioinformatics 30:2114-20 (2014)) to remove adaptors,trim low-quality ends of reads (reads are cut as soon as the basequality dropped below 20 on a 4 bp window), and discard short reads (<50bp). The reads from each sample are then assembled using Idba_ud (Penget al., Bioinformatics 28(11):1420-8(2012)) with kmer size varying from20 to 100 by increment of 10.

The assembled contigs are screened with VirSorter (Roux et al., 2015) toidentify and remove all microbial genomes sequences (i.e. allcontigs >10 kb and not detected as viral by VirSorter in “viromedecontamination” mode). Then, a non-redundant dataset of viral contigsis generated by clustering all viral contigs with Cd-hit (Li et al.,Bioinformatics 22:1658-59 (2006), threshold of 95% ANI on 80% of theshortest sequence). This resulted in 4,759 non-redundant viral sequenceslonger than 10 kb.

To determine the viral population relative abundances in the initialsamples, the QC reads are mapped back to this non-redundant contigsdatabase with bowtie2 (Langmead et al., Nature Methods 9:357-U354(2012), option-non-deterministic and -sensitive, default otherwise). Acontig is considered as detected in a sample if covered by reads on morethan 75% of its length, and its abundance is computed as the contigaverage coverage (number of bp mapped to the contig divided by contiglength) normalized by the total number of bp sequenced in the metagenome(as in Brum et al., Science 348, doi:10.1126/science.1261498 (2015)).

The diversity indices, Shannon's H' and Peilou's J, and Bray-Curtisdistances analyses are calculated using the vegan package (Oksanen etal., Community ecology package, version 2 (2013)) in R version 3.2.3 (RCore Team, 2015). Bray-Curtis distances are statistically ordinatedusing the nonmetric multidimensional scaling (NMDS) and then theinfluence of the metadata on sample ordination is evaluated using the“envfit” function with a total of 9999 permutations in the veganpackage.

Example 17: Long-Term Trial Outcome and Analysis

Clinically, this study remains broadly successful over a two year periodof time. All ASD participants are further assessed for continuedimprovements during a 2 year follow-up. Specifically, overall GIsymptoms, as assessed by the Gastrointestinal Symptom Rating Scale(GSRS), remain significantly improved when assessed at a 2 yearfollow-up (comparing approximately 2.74 average GRS score at thebeginning to approximately 1.72 average GSRS score at the 2 yearfollow-up) (FIG. 23, panel a; and panel b (showing individual patientscores)). The percent change of average GSRS score is 82, 77, and 59%for week 10, week 18, and 2-year follow-up, respectively. GSRS scoresremain improved for subscales including abdominal pain, indigestion,diarrhea, and constipation, (FIG. 23, panel c). The two-year results donot include reflux since no children had significant reflux at the startof the study. Notably, two seemingly opposite GI symptoms—diarrhea andconstipation—continues to respond two years after MTT treatment.

Similarly, the Daily Stool Record (DSR), shows significant decreases inthe number of days with abnormal or no stools at week 10, and theseimprovements remain after 8 weeks of no treatment (Table 7, column 3;and FIG. 24), and at the 2-year follow-up (Table 6, column 9; FIG. 24.Further, significant decreases in constipation is seen at week 10, after8 weeks of no treatment, and at the 2-year follow-up. The Daily StoolRecord (DSR) is collected and averaged over two weeks in order to assesschanges in stool as described in Example 11. Table 7 and FIG. 24 showchanges in stool for initial, week 10, after 8 weeks of no treatment,and during the 2-year follow-up. Overall, participant's showimprovements in defecation.

TABLE 7 Percent days of no stool, constipation, stool hardness andsoftness based on the daily stool record (DSR). Change Change ChangeInitial to Initial to 2-year Initial to Initial Week 10 Week 10 P-ValueWeek 17 Week 17 P-Value Follow Up Follow Up P-Value Hard stool 16%  6%−10% 0.03  3% −13% 0.005  9%  −8% 0.03 (type 1 or 2) Soft/liquid 11%  2% −9% 0.04  3%  −8% 0.05  6%  −5% 0.2 stool (type 6 or 7) No stool 35%25% −10% 0.06 26%  −9% 0.13 27%  −8% 0.2 constipated 52% 31% −21% 0.00729% −22% 0.01 36% −16% 0.06 Abnormal 63% 33% −29% 0.0005 32% −30% 0.00142% −20% 0.009 stool (in total of hard, soft/ liquid/, no stool) Type 1or 2 & 27%  8% −19% 0.004  7% −21% 0.001 15% −12% 0.02 Type 6 or 7

Beyond these GI improvements, ASD-related behavior also improvefollowing MTT. First, the Parent Global Impressions (PGI-R) assessment,which evaluates 17 ASD-related symptoms, reveal significant improvementduring treatment and no reversion 8 weeks after treatment ended or atthe 2-year follow-up (FIG. 25, panel a, showing independent patientresults; and panel b showing average of all ASD patients).

Second, the Vineland Adaptive Behavior Scale II (VABS-II) scoring showsthat the average developmental age increases across 9 of the 11sub-domain areas (FIG. 26, panel a) after 8 weeks of no treatment andthe 2-year follow-up. Among 11 subscales, Fine and Gross Motor skillsare excluded, since these two subscales for the Vineland are onlycalculated up to 6.8 years and most children with ASD improved near tothe limit of the scale. The other 9 subscales and their average arecompared between the baseline and at the end of the study. Significantimprovements are also observed in all 9 subscale areas, at the 2-yearfollow-up, with the largest gains in Interpersonal Skills (3.3 years),Personal Living Skills (2.45 years), and Coping Skills (2.3 years) (FIG.26, panel a). It is notable that the major impairments in ASD, namelyReceptive language, Expressive language, and Interpersonal skills, areamong the lowest initial scores, with initial developmental ages of 3.3years, 4.7 years, and 3.04 years, respectively; all three areas hadsubstantial improvements of 2.6, 0.8, and 3.3 years, respectively. TheVABS-II scoring also shows an overall increase in the averagedevelopmental age by 1.5 years (p<0.001, VABS-II) after 8 weeks with notreatment and 2.3 years (p<0.001, VABS-II), for the 2-year follow-up(FIG. 26, panel b).

Third, ASD-afflicted children show improvements in their scores in theSocial Responsiveness Scale (SRS), which assesses social skill deficitsincluding awareness, cognition, communication, motivation, andmannerisms (FIG. 27, panel a and panel b). ASD-afflicted children alsoshow improvements in their scores in the Aberrant Behavior Checklist(ABC), which evaluates irritability, hyperactivity, lethargy,stereotypy, and inappropriate speech (FIG. 28, panel a). In all fivesub-scales, a significant reduction at the end of treatment is observed.FIG. 28, panel b, shows an overall improvement in ABC scores at week 10,after 8 weeks of no treatment, and at the 2-year follow-up.

Fourth, the Childhood Autism Rating Scale (CARS), which rates core ASDsymptoms, decrease from the severe symptoms range at the beginning ofthe treatment, to the mild-to-moderate symptoms range after 8 weeks ofno treatment and close to the minimal-to-no symptoms range (with someparticipants within the minimal-to-no symptoms range) at the 2-yearfollow-up (p<0.001, FIG. 29, panel a). The percentage of participantswith severe symptoms decrease overtime and the percentage ofparticipants with minimal-to-no symptoms increase overtime (17% and 44%of participants with minimal-to-no symptoms at the end of treatment andat the 2-year follow-up, respectively) (FIG. 29, panel b). Further, asignificant correlation between GSRS and CARS suggests that GI symptomsimpact ASD behaviors, and that these can be altered via MTT (FIG. 29,panels c and d)

Together these findings show that MTT is well-tolerated across anage-diverse cohort of 18 ASD-afflicted children. MTT is also effectiveas it led to significant improvements in GI-symptoms that are mostlysustained at least 2-years after treatment, and improvements inbehavior-related symptoms by the end of treatment, with greaterimprovements at the two-year follow-up.

Example 18: Microbiome Analysis Across Common Diversity Metrics Up tothe 2-Year Follow-Up

Parallel analyses are performed for multiple diversity metrics, asidentified in Examples 12 and 14 above, to understand the effect ofmetric on the microbiome findings. The Observed OTUs metric shows acount of the number of OTUs observed at least two times in a sample(FIG. 30, panel a). FIG. 30, panel b, describes Faith's PhylogeneticDiversity (PD) changes in ASD participants receiving MTT. Similar to theresults of the Observed OTUs, microbial bacterial diversity increased instools of children with ASD, and remained higher than baseline 2-yearsafter the treatment (Follow-up). This confirms that both phylogeneticand non-phylogenetic metrics illustrate the same patterns of change incommunity richness with MTT for at least 2-years.

The Observed OTUs at the 2-years follow-up are also analyzed based onroute of administration, oral vs. rectal. Mann-whitney tests show amarginally higher diversity in participants who receive oraladministration (2-tailed p=0.09), compared to patients receiving MTTrectally (FIG. 30, panel c). No significant difference is observedbetween male and female participants at the 2-year follow-up (FIG. 30,panel d).

Example 18: MTT Changes and Maintains the Abundance of SelectedBacterial Genera in ASD Patients for at Least 2-Years

Several bacterial genera maintain their increase in abundance comparedto the initial treatment when assessed at the 2-year follow-up. Thesegenera include Bifidobacterium (FIG. 31, panel a), Prevotella (FIG. 31,panel b) and Desulfovibrio (FIG. 31, panel c) which all maintainingtheir abundance above the Neurotypical participant median. As outlinedabove in Example 13 (and FIG. 31, panel a and b), Bifidobacterium, andPrevotella are both under-represented in ASD-afflicted children, but atthe 2-year follow-up to MTT, Bifidobacterium significantly increases4-fold (FIG. 30, panel a; p<0.01), and Prevotella significantlyincreases 84-fold (FIG. 31, panel b; p<0.05). These genera also show asignificant increase in abundance at weeks 3 and 10 of treatment, andafter 8 weeks of no treatment. Desulfovibrio also increases 68-foldafter MTT from baseline to the 2-year follow-up. This suggests strongengraftment by these microbes in particular.

Without being bound to any scientific theory, this suggests strongengraftment by these microbes in particular. Bifidobacterium,Desulfovibrio and Prevotella are on average more abundant in MTTrecipients at 2-years following treatment than the neurotypical median,illustrating that the transferred microbiota adapts in its new host, butalso that MTT changes the gut environment in a way that is morehospitable to recruit new commensal bacteria. Taken together, these datasuggest that MTT successfully shifts the ASD microbiota toward that ofage/gender matched neurotypical controls.

The invention claimed is:
 1. A method for increasing the relativeabundance of gut microorganisms in a subject having an autism spectrumdisorder (ASD), said method comprising treating said subject byadministering a therapeutically effective amount of a pharmaceuticalcomposition comprising a fecal microbe preparation comprising acommunity of fecal bacteria from a stool of a human donor, therebyimproving one or more ASD symptoms in said subject and causing saidsubject to exhibit at least a 2-fold increase of the relative abundanceof each of Bifidobacterium and Desulfovibrio in the gut of said subjectafter said treatment as compared to before initiating said treatment. 2.The method of claim 1, wherein said method further comprises determiningin said subject the relative abundance of said Bifidobacterium andDesulfovibrio.
 3. The method of claim 1, wherein said one or more ASDsymptoms comprise an impairment in sociability.
 4. The method of claim1, wherein said subject exhibits at least a 10% reduction in ASD symptomseverity after said treatment as compared to before initiating saidtreatment, and based on an assessment system selected from the groupconsisting of Childhood Autism Rating Scale (CARS), Childhood AutismRating Scale 2-Standard Form (CARS2-ST), Childhood Autism Rating Scale2-High Functioning (CARS2-HF), Aberrant Behavior Checklist (ABC), SocialResponsiveness Scale (SRS), and Vineland Adaptive Behavior Scale II(VABS-II).
 5. The method of claim 1, where said subject exhibits the atleast 2-fold increase after 2 or more weeks of initiating saidadministering of the therapeutically effective amount of thepharmaceutical composition.
 6. The method of claim 1, where said subjectexhibits the at least 2-fold increase for at least 8 weeks afterdiscontinuing said administering of the therapeutically effective amountof the pharmaceutical composition.
 7. The method of claim 1, where saidsubject further exhibits one or more GI symptoms prior to initiatingsaid treatment.
 8. The method of claim 7, where said subject exhibits atleast a 50% reduction in GI symptom severity based on theGastrointestinal Symptom Rating Scale (GSRS) after said treatment ascompared to before initiating said treatment.
 9. The method of claim 1,where said treating further comprises administering an antibiotic tosaid subject prior to administering said pharmaceutical composition. 10.The method of claim 1, wherein said pharmaceutical composition isadministered orally.
 11. The method of claim 1, wherein said fecalmicrobe preparation is lyophilized.
 12. The method of claim 1, whereinsaid fecal microbe preparation comprises a non-selected andsubstantially complete fecal microbiota from a single donor.
 13. Themethod of claim 1, wherein said fecal microbe preparation comprises apreparation of viable flora in proportional content that resembles anormal healthy human fecal flora and comprises no antibiotic resistantpopulations.
 14. The method of claim 1, wherein said pharmaceuticalcomposition is formulated as an acid-resistant capsule.
 15. The methodof claim 1, wherein said fecal microbe preparation comprises bacteriafrom the genus Lactobacillus.
 16. The method of claim 15, wherein saidbacteria from the genus Lactobacillus are cultured.
 17. The method ofclaim 16, wherein said fecal microbe preparation further comprises anon-selected fecal microbiota.
 18. The method of claim 1, wherein saidfecal microbe preparation is spray-dried.
 19. The method of claim 1,wherein said pharmaceutical composition is formulated as acid-resistantmicrocapsules.
 20. The method of claim 19, wherein said pharmaceuticalcomposition is administered to the subject with a food or drink.